Methods and compositions for use of recombinant bacterial effector proteins as anti-inflammatory agents

ABSTRACT

Provided herein are methods and compositions comprising a set of paired peptides comprising a first bacterial effector polypeptide or fragment thereof linked to a second bacterial effector polypeptide or fragment thereof. The paired peptides can be linked to a protein transduction domain. The compositions can be formulated as pharmaceuticals. The compositions are useful for the treatment of inflammatory disorders.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for treatinginflammatory disorders.

BACKGROUND OF THE INVENTION

Most treatments for acute inflammation, such as skin inflammatoryconditions, only treat the symptoms (swelling, redness, pain, heat) byusing non-specific drugs like corticosteroids and emollients/skinsofteners. None of these non-specific drugs affect the underlyingmechanism of activation of the inflammatory pathway, e.g., NFkB/JNK/p38.Moreover, normal aging processes chronically activate ectopically thesethree signal transduction pathways, resulting in inflammation. Theinflammation makes these pathways a target for inhibition as part of ananti-aging/wellness program.

To date, the cosmeceutical industry's approach to reducing skininflammation has many flaws. The “Active Compounds” contained in mostcremes and topicals do not have defined, specific targets in theinflamed cell/tissue the cell and are extremely complexmixtures/extracts/serums containing millions of ingredients with nodefined targets, or ingredients which are not absorbed by skin and haveno effect at all. Because these compositions do not target the actualpathways which are causing inflammation, they have the potential toalter many processes non-specifically leading to toxic side effects.

A continuing need in the art exists for new and effective tools andmethods for treating the causes of inflammation.

SUMMARY OF THE INVENTION

Disclosed herein are compositions of paired peptides comprising a firstbacterial effector polypeptide linked to a second bacterial effectorpolypeptide that are useful for treating an inflammatory disorder.Accordingly, disclosed are compositions that include a set of pairedpeptides, wherein the set of paired peptides is linked to a proteintransduction domain, and wherein the set of paired peptides comprises afirst bacterial effector polypeptide or fragment thereof linked to asecond bacterial effector polypeptide or fragment thereof. The first andsecond bacterial effector polypeptides can be different, that is theycan recognize a different molecular targets or modulate differentinflammatory pathways. In an embodiment, the protein transduction domainand the set of paired peptides can be a fusion protein. The fusionprotein can include one or more linkers. The protein transduction domaincan be a YopM protein transduction domain, an SspH1 protein transductiondomain, or an IpaH protein transduction domain. The first bacterialeffector polypeptide or fragment thereof can be a polypeptide selectedfrom the group consisting of NleE, NleC, NleD, NleB, NleH, YopM, YopE,YopH, YopJ, YopP, SspH1, OspG, OspF, IpaH9.8, IpaH1.4, IpaH2.5, IpaH4.5,IpaH7.8 and SIrP, and the second bacterial effector polypeptide orfragment thereof can be a polypeptide selected from the group consistingof NleE, NleC, NleD, NleB, NleH, YopM, YopE, YopH, YopJ, YopP, SspH1,OspG, OspF, IpaH9.8, IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8 and SIrP. Insome embodiments, the first bacterial effector polypeptide or fragmentthereof can be a polypeptide having 90% sequence identity to an aminoacid sequence set forth in the group consisting of SEQ ID NOs 3, 89, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,and 79 and the second bacterial effector polypeptide or fragment thereofcan be a polypeptide having 90% sequence identity to an amino acidsequence set forth in the group consisting of SEQ ID NOs.3, 89, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and79. In some embodiments, the first bacterial effector polypeptide orfragment thereof can be a YopM polypeptide or a fragment thereof and thesecond bacterial effector polypeptide or fragment thereof can be an NLeEpolypeptide or a fragment thereof. In some embodiments, the fusionprotein can have an amino acid sequence forth in SEQ ID NO. 10, 13, 16,19, 22, or 24.

Also provided¶ are fusion proteins comprising a set of paired peptideswherein the set of paired peptides comprises a first bacterial effectorpolypeptide or fragment thereof linked to a second bacterial effectorpolypeptide or fragment thereof. The first and second bacterial effectorpolypeptides can be different, that is they can recognize a differentmolecular targets or modulate different inflammatory pathways. Thefusion protein can include one or more linkers. The first bacterialeffector polypeptide or fragment thereof can be a polypeptide selectedfrom the group consisting of NleE, NleC, NleD, NleB, NleH, YopM, YopE,YopH, YopJ, YopP, SspH1, OspG, OspF, IpaH9.8, IpaH1.4, IpaH2.5, IpaH4.5,IpaH7.8 and SIrP, and the second bacterial effector polypeptide orfragment thereof can be a polypeptide selected from the group consistingof NleE, NleC, NleD, NleB, NleH, YopM, YopE, YopH, YopJ, YopP, SspH1,OspG, OspF, IpaH9.8, IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8 and SIrP. Insome embodiments, the first bacterial effector polypeptide or fragmentthereof can be a polypeptide having 90% sequence identity to an aminoacid sequence set forth in the group consisting of SEQ ID NOs 3, 89, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,and 79 and the second bacterial effector polypeptide or fragment thereofcan be a polypeptide having 90% sequence identity to an amino acidsequence set forth in the group consisting of SEQ ID NOs.3, 89, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and79. In some embodiments, the first bacterial effector polypeptide orfragment thereof can be a YopM polypeptide or a fragment thereof and thesecond bacterial effector polypeptide or fragment thereof can be an NLeEpolypeptide or a fragment thereof. In some embodiments, the fusionprotein comprising a set of paired peptides can have an amino acidsequence as set forth in SEQ ID NO. 80, 81, 82, 83, 84, 85, 86, 87, or88.

Also provided are nucleic acids encoding a set of paired peptides,wherein the set of paired peptides is linked to a protein transductiondomain, and wherein the set of paired peptides comprises a firstbacterial effector polypeptide or fragment thereof linked to a secondbacterial effector polypeptide or fragment thereof. Also provided arenucleic acids encoding fusion proteins comprising a set of pairedpeptides wherein the set of paired peptides comprises a first bacterialeffector polypeptide or fragment thereof linked to a second bacterialeffector polypeptide or fragment thereof. The nucleic acids can becontained within a vector, which can be expressed in a host cell. In oneaspect, the compositions comprising a set of paired peptides can beformulated as pharmaceutical compositions.

Also provided are methods of treating a subject having or at risk for aninflammatory disorder, by administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising the set ofpaired peptides. The inflammatory disorder can be a gastrointestinaldisorder including inflammatory bowel disease, Crohn's disease and theileocolitis, ileocecal, jeunoileitis, and gastroduodenal subtypesof-Crohn's disease, and ulcerative colitis. The inflammatory disordercan also be a skin disorder.

Also provided are articles of manufacture, e.g., a kit. The kit caninclude measured amount of one or more of the compositions of the pairedpeptides and one or more items selected from the group consisting ofpackaging material, a package insert comprising instructions for use, asterile fluid, and a sterile container.

In one aspect, a composition comprises in a pharmaceutically acceptablecarrier or excipient or formulation a first construct comprising aselected immunomodulatory effector protein or functional equivalentthereof that targets a first functional domain, optionally linkedcovalently or non-covalently to a selected protein transduction domain(PTD) or penetrating peptide (CPP). In one embodiment, the compositionfurther comprises an additional construct comprising a differenteffector protein or a functional equivalent thereof that targets anadditional functional domain, optionally linked to the same PTD or CPPor to an additional PTD or CPP. In one embodiment, a compositioncomprises multiple first and additional constructs. In anotherembodiment, the constructs are further associated with targetingmoieties directing delivery of the constructs to a selected cell ortissue.

In another aspect, a recombinant polypeptide comprises a first constructcomprising a selected immunomodulatory effector protein or functionalequivalent thereof that targets a first functional domain, optionallylinked covalently or non-covalently to a selected protein transductiondomain (PTD) or penetrating peptide (CPP) and an additional constructcomprising a different effector protein or a functional equivalentthereof that targets an additional functional domain, optionally linkedto the same PTD or CPP or to an additional PTD or CPP. The firstconstruct is linked covalently or non-covalently to one or more of theadditional constructs in a single polypeptide. In another embodiment,the polypeptide comprises an optional linker amino acid sequenceinterposed between each first and additional construct. In anotherembodiment, the polypeptide is further associated with targetingmoieties directing delivery of the polypeptide to a selected cell ortissue.

In another aspect, a recombinant nucleic acid molecule is provided whichencodes one of the constructs or polypeptides described herein. Thesenucleic acid molecules can be further associated with regulatorysequences for expressing the constructs in vivo or in vitro.

In a further aspect, a pharmaceutical or cosmeceutical compositioncomprises as an active agent a polypeptide as described above, or amixture of constructs as described above in a formulation suitable fordelivery of the active agent into and through the layers of the skin. Inone embodiment, the formulation contains a CAGE solvent (defined below)or other components suitable for topical administration.

In yet another aspect, a pharmaceutical or cosmeceutical compositioncomprises as an active agent a polypeptide as described above, or amixture of constructs as described above in a formulation suitable fordelivery to a selected cell or tissue.

In still other aspects, methods for making the compositions, constructs,polypeptides and nucleic acid molecules are provided.

In yet a further aspect, a method for treating or ameliorating orsuppressing an inflammatory response comprises administering to asubject in need thereof a composition, construct, polypeptide or nucleicacid molecule described herein.

Still other aspects and advantages of these compositions and methods aredescribed further in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the present invention will bemore fully disclosed in, or rendered obvious by, the following detaileddescription of the preferred embodiment of the invention, which is to beconsidered together with the accompanying drawings wherein like numbersrefer to like parts and further wherein:

FIG. 1 is a schematic of bacterial effector constructs.

FIG. 2 is a schematic of bacterial effector constructs.

FIG. 3 is a schematic of the cloning strategy for TAT-NleE WT and MutantR107A.

FIG. 4 is a schematic of the cloning strategy for TAT-Shigella OSPZ.

FIG. 5 is a schematic of the cloning strategy for YopM PTD-NleE WT andMutant R107A.

FIG. 6 is a schematic of the cloning strategy for YopM PTD-NleE WT NoLinker, PAPA Linker, GSGS Linker and for Mutant R107A.

FIG. 7 is an exemplary SDS gel showing purified fusion proteins.

FIG. 8 is a graph showing the results of a dose-response analysis ofIL-6 production in cells treated with recombinant purified effectorproteins.

FIG. 9 is a graph showing the results of an analysis of the effect ofpaired fusion proteins on TNF-alpha.

FIG. 10 is a graph showing the results of an analysis of the effect ofpaired fusion proteins on IL-6.

FIG. 11 is a graph showing the results of an analysis of the effect ofpaired fusion proteins on MCP-1.

FIG. 12 is a graph showing the results of an analysis of the effect ofpaired fusion proteins on IL-23.

FIG. 13 is 12 is a graph showing the results of a dose response analysisof rYopM and YopMo on caspase 1 activity.

FIG. 14 shows the uptake of FITC-TAT-NleE-WT-His protein by Hacat cells.

FIG. 15 shows the uptake of FITC-YopM PTD-YopM (L-Rich)-GSGS LinkerNleE-WT-His protein by Hacat cells.

FIG. 16 shows the uptake of FITC-YopM PTD-YopM (L-Rich)-PAPALinker-NleE-WT-His protein by Hacat cells

FIG. 17 shows the uptake of FITC-YopM PTD-YopM (L-Rich)-GSGSLinker-NleE-WT-His protein by Hacat cells.

FIG. 18 shows the uptake of TAT-NleE fusion polypeptide into intactmouse skin.

FIG. 19 shows is an two-photon microscopy image of uptake of TAT-NleEfusion polypeptide into intact mouse skin.

FIG. 20 shows a two-photon microscopy image of a 10 micron slice ofmouse skin showing uptake of TAT-NleE fusion polypeptide into intactmouse skin.

FIG. 21 shows graphs illustrating an analysis of methylase activity ofYopM PTD-YopM (L-Rich)-PAPA/GSGS-NleE-WT-His fusion proteins.

FIG. 22 shows graphs illustrating an analysis of methyltransferaseactivity NleE-R107A mutant and YopM.

FIG. 23 shows graphs illustrating a NleE and Shigella Methyltransferaseactivity assay.

FIG. 24 depicts the results of an experiment comparing the effect offormulations on NleE methylation activity.

FIG. 25 shows four micrograph panels showing transdermal penetration ofCAGE-NleE protein.

FIG. 26 is a schematic showing how inflammation develops from cell totissue in the skin.

FIG. 27 is a schematic showing the impact of a skin irritant on the NFKBpathways.

FIG. 28 shows the amino acid sequence of NleE SEQ ID NO: 1.

FIG. 29 shows the publicly available UniProt P17778 amino acid sequenceof YopM SEQ ID NO: 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

This description of preferred embodiments is intended to be read inconnection with the accompanying drawings, which are to be consideredpart of the entire written description of this invention. The drawingfigures are not necessarily to scale and certain features of theinvention may be shown exaggerated in scale or in somewhat schematicform in the interest of clarity and conciseness. In the description,relative terms such as “horizontal,” “vertical,” “up,” “down,” “top” and“bottom” as well as derivatives thereof (e.g., “horizontally,”“downwardly,” “upwardly,” etc.) should be construed to refer to theorientation as then described or as shown in the drawing figure underdiscussion. These relative terms are for convenience of description andnormally are not intended to require a particular orientation. Termsincluding “inwardly” versus “outwardly,” “longitudinal” versus “lateral”and the like are to be interpreted relative to one another or relativeto an axis of elongation, or an axis or center of rotation, asappropriate. Terms concerning attachments, coupling and the like, suchas “connected” and “interconnected,” refer to a relationship whereinstructures are secured or attached to one another either directly orindirectly through intervening structures, as well as both movable orrigid attachments or relationships, unless expressly describedotherwise. The term “operatively connected” is such an attachment,coupling or connection that allows the pertinent structures to operateas intended by virtue of that relationship. When only a single machineis illustrated, the term “machine” shall also be taken to include anycollection of machines that individually or jointly execute a set (ormultiple sets) of instructions to perform any one or more of themethodologies discussed herein. In the claims, means-plus-functionclauses, if used, are intended to cover the structures described,suggested, or rendered obvious by the written description or drawingsfor performing the recited function, including not only structuralequivalents but also equivalent structures.

Technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs and by reference to published texts, which provide oneskilled in the art with a general guide to many of the terms used in thepresent application. The definitions contained in this specification areprovided for clarity in describing the components and compositionsherein and are not intended to limit the claimed invention.

The present invention is based in part on the inventors' finding thatcombinations of bacterial effector polypeptides can have synergisticimmunomodulatory activity. Many bacterial pathogens, includingenteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli, andShigella, utilize a type III secretion system (T3SS) to deliver multiplevirulence proteins directly into host cells. These virulence proteins,also referred to as effector proteins, are produced by bacteria duringthe infection of a eukaryotic host. The effector proteins down-regulatethe host's immune system, typically at the site of the infection. Thus,effector proteins that target specific inflammatory pathways canfunction as immunomodulators, for example, in the treatment ofinflammatory disorders. The inventors have found that combinations ofeffector proteins that target multiple inflammatory pathways producedaugmented immunomodulatory effects.

Accordingly, the invention features compositions and methods that areuseful for the treatment of inflammatory disorders. The compositions caninclude fusion proteins comprising bacterial effector polypeptides or afragment of a bacterial effector polypeptide. More specifically, thecompositions can include a set of paired peptides configured as a firstbacterial effector polypeptide or a fragment thereof and a secondbacterial effector polypeptides or a fragment thereof. For ease ofreading, we will not repeat the phrase “or a fragment thereof” on everyoccasion. It is to be understood that where we refer to a firstbacterial effector polypeptide, we refer to the first bacterial like andeffector polypeptide or a fragment thereof. Similarly, it is to beunderstood that where we refer to a second bacterial effectorpolypeptide, we refer to the second bacterial effector polypeptide or afragment thereof. Also featured are compositions comprising a set ofpaired peptides configured as a first bacterial effector polypeptide ora fragment thereof and a second bacterial effector polypeptides or afragment thereof, and linked to a protein transduction domain. Themethods can include a method of treating a subject at risk for or havingan inflammatory disorder.

The compositions and methods described herein deliver a combinatorialconstruct of small recombinant protein effectors that directly targetcellular pathways involved in inflammation, e.g., the NFKB pathwayactivated in skin inflammation. As described in detail below, thedelivery of a recombinant construct comprising one or multiple effectorproteins linked or fused to a PTD or CPP is useful to treatinflammation. Still other embodiments include the association of anoptional targeting moiety, directing the construct to a specific cell ortissue type, with the effector protein and/or formulation in a topicalcarrier. While these compositions and methods of treatment have a numberof advantages, a significant advantage is that the protein effectorsused are not able to enter into the circulation. In one embodiment, asdiscussed in detail below, the compositions and methods involve the useof NleE incorporated into a cream or oil formulation that reduces skininflammation and may be widely used for many applications, both as acosmetic beauty crème to reduce redness and irritation and as atreatment of a disease or cause of skin irritation.

The present compositions and methods using multiple e.g., NFkB/MAPKinhibitors, for treatment of inflammatory responses (e.g., pain,redness, swelling, heat) is based upon the fact that inflammation causedby infection, injury, auto-immunity, sunburn, aging, etc is detected atthe cell membrane. Signals that are received by the skin cell arefunneled thru the NFKB (sometimes MAPK) pathway. This is a relaymechanism in the cell that must be highly regulated. Shutting off theNFKB pathway is the key to controlling skin inflammation. Naturallyoccurring bacterial effector proteins (e.g., NleE, YopM, SSPH1) are verypotent inhibitors of the NFKB system. Their sole target and purpose isto dampen the inflammatory response.

As described herein, when these bacterial proteins are recombinantlyengineered into a selected polypeptide and delivered to inflamed skinvia fusion with a PTD/CPP and optional targeting moiety, they abolishthe inflammatory response. The combination of two or more effectorproteins, each with a different substrate in the cell, may becombinatorially fused together in a single polypeptide. In someembodiments, such combination achieves a synergistic effect, which is asignificant improvement in activity beyond that accomplished by deliveryof a single effector.

The bacterial effector, NleE, is characterized by specificity, potencyand efficiency in shutting down NFkB and hence inflammatory reactions.The presence of NleE and/or a combination with other bacterial effectorsin an anti-inflammatory formulation has a number of advantages. Amongthe advantages are extremely high substrate specificity, which resultsin only a very low, if any, chance of off-target effects and toxicityfor therapeutic use. In one embodiment, fusing PTDs to NleE and itsrelated effectors is useful to deliver the effector to sites ofinflammation. We and others have shown that a number of recombinanteffector proteins, when fused to PTDs can indeed cross the cell andtissue boundary and be taken up by cells resulting in NFkB/JNK/p38pathway inhibition

The methods and compositions described below provide combinations ofmultiple effectors, or single or multiple effector(s) fused with a PTD,or single or multiple effector(s) fused with a targeting moiety, orsingle or multiple effector(s) fused with a PTD and a targeting moiety,in a chimeric recombinant protein, along with an emollient compound(such as CAGE) for use as a topical anti-inflammatory crème for manydifferent ailments.

Compositions

Provided herein are compositions comprising engineered bacterialeffector polypeptides for use in the treatment of inflammation. Theengineered bacterial effector polypeptides can be configured as a set ofpaired peptides. More specifically, a set of paired peptides can be aconstruct comprising a first bacterial effector polypeptide or afragment thereof and a second bacterial effector polypeptides or afragment thereof. The set of paired peptides can be linked to one ormore polypeptide sequences that facilitate intracellular delivery of thepaired peptides, for example, a protein transduction domain (PTD) or acell penetrating peptide (CPP).

Bacterial effector polypeptides. The first bacterial effectorpolypeptide and the second bacterial effector polypeptide can be abacterial effector polypeptide selected from the exemplary bacterialeffector polypeptides from a variety of bacteria as shown in Tables 1and 2. Their enzymatic activity and host targets are also shown inTables 1 and 2. Representative Uniprot or Genbank references for thepolypeptides are shown in Tables 3 and 4. Additional amino acidsequences for, and nucleic acid sequences encoding, these bacterialeffector polypeptides can be identified from databases such as UniProt,NCBI, GenBank and publications extant in the art.

TABLE 1 BACTERIAL T3SS EFFECTORS Effector Bacteria IntracellularActivity Host Target OspF Shigella Phosphothreonine lyase ERK, p38flexneri MAPKs OspG Shigella Serine/threonine kinase E2 ubiquitinflexneri ligases NIeH1 EPEC-EHEC Serine/threonine kinase RPS3 NIeE/OspZEPEC-EHEC/ Cysteine methylase TAB2/NfKB Shigella NIeB EPEC-EHEC O-GIcNActransferase FADD, GAPDH, RIPK1, TRADD NIeC EPEC-EHEC Zincmetalloprotease NFkB YopH Yersinia Phosphotyrosine Akt/FAK phosphataseYopE Yersinia Rho GAP Rho GTPases/ caspases YopP/YopJ YersiniaAcetyltransferase MAP Ks YopM Yersinia LRR motif PKN/RSK

TABLE 2 BACTERIAL T3SS E3 UBIQUITIN LIGASE EFFECTORS E3 Ligase FactorType/ Family Factor Bacteria Intracellular Activity Host Target HECTSopA Salmonella Regulation of host TRIM65/56 typhimurium inflammationNIeL EPEC/EHEC Formation of actin Unknown pedestal RING NIeG EPEC/EHECUnknown Unknown U-Box LubX Legionella Regulation of another Cdh1, SidHpneumophila effector function Gob X L. pneumophila Unknown Unknown NELIpaH1.4 Shigella Inhibition of NF-Kb HOIP flexneri activation IpaH2.5 S.flexneri Inhibition of NF-kB HOIP activation IaH4.5 S. flexneriInhibition of NF-kB p65, TBK1 and I-IFN activation IpaH7.8 S. flexneriInduction of GLMN pyroptosis IpaH9.8 S. flexneri Inhibition of NF-kBNEMO activation IpaH0722 S. flexneri Inhibition of NF-kB TRAF2activation SspH1 S. typhimurium Inhibition of androgen PKN1 receptorSspH2 S. typhimurium Promotion of IL-8 Nodi, SGT1 secretion SIrP S.typhimurium Induction of host cell Trx death SidE SidC L. pneumophilaUnknown Unknown family (SdcA)

TABLE 3 Representative Amino Acid Sequences of Bacterial T355 EffectorsUniProt or Genbank Effector Bacteria Host Target Reference ID OspFShigella ERK, p38 Q8VSP9 flexneri MAPKs (OSPF_SHIFL) OspG Shigella E2ubiquitin Q99PZ6 flexneri ligases (OSPG_SHIFL) NIeH1 EPEC- RPS3 Q8X831EHEC (Q8X831_ECO57) NIeE/ EPEC- TAB2/NfKB Q7DBA6 OspZ EHEC/(Q7DBA6_ECO57) Shigella NIeB EPEC- FADD, GAPDH, VEC94465.1 EHEC RIPK1,TRADD (Genbank) NIeC EPEC- NFkB CBG88408.1 EHEC (Genbank) YopH YersiniaAkt/FAK P15273 (YOPH_YEREN) YopE Yersinia Rho P31492 GTPases/caspases(YOPE_YEREN) YopP/YopJ Yersinia MAPKs O34336 (YOPP_BACSU) YopM YersiniaPKN/RSK P17778 (YOPM_YERPE)

TABLE 4 Representative Amino Acid Sequences of Bacterial T3ss E3Ubiquitin Ligase Effectors E3 Ligase Factor UniProt or GenbankType/Family Factor Bacteria Host Target Reference ID HECT SopASalmonella TRIM65/56 Q8ZNR3 typhimurium (SOPA_SALTY) NIeL EPEC/EHECUnknown A0A0D6ZN92 (A0A0D6ZN92_ECOLX) RING NIeG EPEC/EHEC UnknownA0A023YUN6 (A0A023YUN6_ECOLX) U-Box LubX Legionella Cdh1, Q5ZRQ0pneumophila SidH (LUBX_LEGPH) Gob X L. pneumophila Unknown NEL IpaH1.4Shigella HOIP A0A380D014 flexneri (A0A380D014_SHIFL) IpaH2.5 S. flexneriHOIP Q99Q42 (Q99Q42_SHIFL) IaH4.5 S. flexneri p65, TBK1 P18009(IPA4_SHIFL) IpaH7.8 S. flexneri GLMN P18014 (IPA7_SHIFL) IpaH9.8 S.flexneri NEMO Q8VSC3 (IPA9_SHIFL) IpaH0722 S. flexneri TRAF2 SspH1 S.typhimurium PKN1 D0ZVG2 (SSPH1_SALT1) SspH2 S. typhimurium Nod1, P0CE12SGT1 (SSPH2_SALTY) SIrP S. typhimurium Trx Q8ZQQ2 (SLRP_SALTY) SidEfamily SidC L. pneumophila Unknown Q6RCR3 (SdcA) (Q6RCR3_LEGPN)

Thus a bacterial effector peptide can be an SspH1; SspH2; SIrP; IpaH1.4;IpaH2.5; IpaH3; IpaH4.5; IpaH7.8; IpaH9.8; NleE; NleC; NleD; NleB; NleH;NleH1; YopM; YopE; YopH; YopJ; YopP; OspG; OspF; OspZ; OspI; SopE; SopB;SopE2; SipA; AvrA; SseL; EspT; or a TiR polypeptide.

In some embodiments, the bacterial effector polypeptide can have anamino acid sequence at least 90% identical to an amino acid sequence setforth in any of SEQ ID NOs. 3, 89, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 79. In some embodiments, thebacterial effector polypeptide have an amino acid sequence as set forthin any of SEQ ID NOs. 3, 89, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, and 79.

The term “type III secretion system or T355” refers to a highlyspecialized molecular needle construct containing a Yersiniaeinjectisome spanning the bacterial membranes, Yersinia outer protein(Yop) effectors and Yop translocators needed to deliver the effectorsacross the membrane (Camelis G R., Int J Med Microbial. 2002 February;291(6-7):455-62). Pathogenic Yersiniae require this T3SS to survive andreplicate extracellularly within lymphoid tissues of their animal orhuman hosts. See, also, U.S. Pat. No. 8,840,901.

The term “immodulatory effector protein” refers to small proteins,generally bacterial in origin, that suppress the human innate immunesystem during infection. These effector proteins activate the NFkB, JNK,and p38 signaling pathways during infection. These effector proteins areoften secreted into the cells targeted for infection by T3SS. Onceinside the cell, each effector protein targets a single host proteinrequired for innate immunity, which it inactivates using a myriad ofmechanisms including acetylation, methylation, action of phosphatases onP04 proteins, induced protein degradation etc.

All T3SS bacterial effector proteins are very small, globular, highlystable, highly catalytic, have high substrate specificity, bindco-factors very tightly, and can be injected into the cell in denaturedform. They modify every substrate molecule in the cell. For instance,the EPEC effector NleE is a cysteine methyltransferase which has asingle target in the cell, i.e., the TAB2 scaffold protein in the NFkBpathway. EPEC are mildly infective gut bacteria that attach to coloncells and directly inject virulence proteins thru a T3SS to controlinnate immune pathways as a survival strategy. During EPEC infection,the injected NleE protein methylates every molecule of cellular TAB2;thereby completely shutting off NFkB signaling, as described in Yao, Q.et al., Structure and Specificity of the Bacterial CysteineMethyltransferase Effector NleE Suggests a Novel Substrate in Human DNARepair Pathway., PLoS Pathogens (November 2014) 10(11):e1004522;doi:10.1371/journal.ppat.1004522.

Still another effector protein is YopM, discussed in U.S. Pat. No.8,840,901 and Rüter, C & Hardwidge, PR, Drugs from Bugs′: bacterialeffector proteins as promising biological (immune-) therapeutics. FEMSMicrobiol Lett 351 (December 2013/January 2014) 126-132.

In some embodiments, the first and second bacterial effectorpolypeptides are different. For example, they can have different aminoacid sequences, different structures, different functions, differentmolecular targets, or have non-overlapping redundant roles in inhibitingan inflammatory pathway, for example, the NFkB, JNK, p38, and STINGpathways.

Also included as effectors or effector proteins of the compositions andmethods described herein are functional equivalents of the proteinsdescribed above. By the term “functional equivalent” is meant any aminoacid sequence or modification thereof that has the same targeting andimmune suppressing function of the naturally occurring effector protein.In one embodiment, such functional equivalents can have modifications ofone or more amino acids from the known sequences. In one embodiment,such functional equivalents can be a smaller fragment of the knownsequences. In one embodiment, such functional equivalents can be aderivative of the naturally occurring sequences or be derived from otherthan human sources. In one embodiment, such functional equivalents canbe altered by chemical modification or be altered by recombinantproduction to be associated with sequences with which the effectorproteins are not associated in nature. Similarly, chemical or structuralchanges or fragments of the nucleic acid sequences that encode theeffector proteins are also considered functional equivalents herein.

The paired peptides can be joined by a linker. A linker can be anyreagent, molecule or macromolecule that connects the first and secondbacterial effector polypeptides such that the linker does notsubstantially alter the physiological activity of the effectorpolypeptides. A linker can be a peptide bond. That is, the first andsecond bacterial effector polypeptides or fragments thereof can be afusion polypeptide comprising one or more amino acid segments from thefirst bacterial effector polypeptide and one or more amino acid segmentsfrom second bacterial effector polypeptide. The term “amino acidsegment” as used herein refers to a contiguous stretch of amino acidswithin a polypeptide. For example, the amino acid residues 30 to 40within a 100 amino acid polypeptide would be considered an amino acidsegment. An amino acid segment can be a length greater than eight aminoacid residues (e.g., greater than about nine, ten, 15, 20, 25, 30, 40,50, 75, 100, 150, 200, 500, 1000, or more amino acid residues). In someembodiments, an amino acid segment can have a length less than 1000amino acid residues (e.g., less than 500, less than 400, less than 350,less than 300, less than 200, or less than 100 amino acid residues). Inother embodiments, an amino acid segment can have a length from about 20to about 200 amino acid residues (e.g., about 30 to about 180 amino acidresidues, or about 40 to about 150 amino acid residues).

The amino acid segments of the first bacterial effector polypeptide canbe contiguous with the amino acid segments of the second or they can beseparated by amino acids inserted as a structural spacer. A spacersegment can be one or more amino acids. The one or more amino acids caninclude amino acids that are the same or that are different. Forexample, a spacer can be a repeating series of a neutral amino acid(e.g., glycine, alanine, valine, isoleucine or leucine) ranging innumber from 1 to 10 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore). Another example of a spacer configuration can be a series ofinterspersed amino acids that may be neutral (e.g.,glycine-alanine-glycine-alanine-glycine-alanine, orglycine-glycine-glycine-valine-valine-valine) or charged amino acids(e.g., glutamate-glutamate-glutamate-arginine-arginine-arginine, oraspartate-lysine-aspartate-lysine-aspartate-lysine) or amino acids withother functional groups (e.g.,proline-proline-proline-serine-serine-serine ortyrosine-glutamine-cysteine-methionine-tryptophan) ranging in numberfrom 1 to 10 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). Inanother embodiment, a spacer configuration can be a sequence of aminoacids derived from a naturally occurring protein such as the hingeregion joining the heavy chain CH1 and CH2 domains of immunoglobulin G.In some embodiments, the linker can be a GSGS linker (SEQ ID NO. ______)or a PAPA linker (SEQ ID NO. ______). In some embodiments, the fusionprotein comprising a set of paired peptides can exclude a linker.

A fusion protein can be produced in vitro by continuous peptidesynthesis according to standard chemical methods know to those in theart. Synthetic polypeptides can also be purchased from commercialsources. A fusion protein can also be produced by recombinant DNAtechniques. Nucleic acid segments encoding the first bacterial effectorpolypeptide can be operably linked in the same open reading frame tonucleic acid sequences encoding the second bacterial effectorpolypeptide in a vector that includes the requisite regulatory elements,e.g., promoter sequences, transcription initiation sequences, andenhancer sequences, for expression in prokaryotic or eukaryotic cells.

The paired peptide constructs can include a combination of any of anSspH1; SspH2; SIrP; IpaH1.4; IpaH2.5; IpaH3; IpaH4.5; IpaH7.8; IpaH9.8;NleE; NleC; NleD; NleB; NleH; NleH1; YopM; YopE; YopH; YopJ; YopP; OspG;OspF; OspZ; OspI; SopE; SopB; SopE2; SipA; AvrA; SseL; EspT; or a TiRpolypeptide. Thus, the the first bacterial effector polypeptide can be apolypeptide selected from the group consisting of NleE, NleC, NleD,NleB, NleH, YopM, YopE, YopH, YopJ, YopP, SspH1, OspG, OspF, IpaH9.8,IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8 and SIrP, and the second bacterialeffector polypeptide can be a polypeptide selected from the groupconsisting of NleE, NleC, NleD, NleB, NleH, YopM, YopE, YopH, YopJ,YopP, SspH1, OspG, OspF, IpaH9.8, IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8 andSIrP.

In some embodiments, the paired peptide construct can include a firstbacterial effector polypeptide or fragment thereof is a YopM polypeptideor a fragment thereof or an NLeE polypeptide or a fragment thereof and asecond bacterial effector polypeptide or fragment thereof is a YopMpolypeptide or a fragment thereof or an NLeE polypeptide or a fragmentthereof. In some embodiments, the first bacterial effector polypeptideor fragment thereof is a YopM polypeptide or a fragment thereof and thesecond bacterial effector polypeptide or fragment thereof is an NLeEpolypeptide or a fragment thereof.

In some embodiments, the paired peptide constructs can be configured assummarized in Table 5 below.

TABLE 5 Paired Peptide Fusion Constructs Construct SEQ ID name Effector1 Linker Effector 2 NO YopM PTD- YopM (L-rich) — EPEC NLeE 80 NIeE (Nolinker) YopM PTD- YopM (L-rich) GSGS EPEC NLeE 82 NIeE (GSGS linker)YopM PTD- YopM (L-rich) PAPA EPEC NLeE 81 NIeE (PAPA linker)

In some embodiments, paired peptide fusion protein can have an aminoacid sequence is at least 85% identical to the sequence set forth in SEQID NO. 80, 81, 82, 83, 84, 85, 86, 87, or 88. In some embodiments,paired peptide fusion protein can have an amino acid sequence is atleast 90% identical to the sequence set forth in SEQ ID NO. 80, 81, 82,83, 84, 85, 86, 87, or 88. In some embodiments, paired peptide fusionprotein can have an amino acid sequence is at least 95% identical to thesequence set forth in SEQ ID NO. 80, 81, 82, 83, 84, 85, 86, 87, or 88.In some embodiments, paired peptide fusion protein can have an aminoacid sequence is at least 99% identical to the sequence set forth in SEQID NO. 80, 81, 82, 83, 84, 85, 86, 87, or 88. In some embodiments, thepaired peptide fusion protein can have an amino acid sequence as setforth in SEQ ID NOs. 80, 81, 82, 83, 84, 85, 86, 87, or 88.

Protein transduction domains. The set of paired peptides can be linkedto one or more polypeptide sequences that facilitate intracellulardelivery of the paired peptide. The terms “protein transduction domain(PTDs)” and/or “cell-penetrating peptide (CPPs)” refers to powerfulsequences that allow intracellular delivery of conjugated cargoes tomodify cell behavior. These small peptides can transport a wide varietyof biologically active conjugates into the cell. Heterologous CPP codingsequences are added to effectors or effector-fusions to facilitatecellular uptake of the proteins into cells and tissues, including use ofendogenous CPPs encoded in native effector proteins. This includesaddition of CPP sequences or modules to the effector via chemicalcrosslinking, attachment to a nano-particle or other scaffold chemicallyor via PPIs for the purpose of transporting the effector across tissueand cell membranes. Among useful PTD or CPPs for the present methods andcompositions are those known and identified in the art, including,without limitation, HIV Tat protein basic domain, (HIV Tat amino acids48-60 or 49-57), poly-Arg or polyLys, penetratin, MPG, Pep-1, MAP, andtransportan. See, e.g., Table 1 of Guidotti, G. et al, Trends inPharmacological Sciences (April 2017), 38(4):406-424, which includesadditional examples of CPPs and sequences origins and properties. OtherCPPs are described in Norkowski, S. et al, Bacterial LPX motif-harboringvirulence factors constitute a species-spanning family ofcell-penetrating effectors, Cellular and Molecular Life Sciences(December 2017) doi.org/10.1007/s00018-017-2733-4, which describedprototypes of such bacteria-derived cell-penetrating effectors (CPEs)including the Yersinia enterocolitica-derived YopM, the Salmonellatyphimurium effector SspH1, and the Shigella IpaH proteins. Still otherprotein transporter molecules include those previously described inDixon, J E et al, Proc. Natl Acad Sci, (January 2016), E291-299; as wellas synthetic protein mimics described by Tezgel, A O et alBioMacromolecules (2017) 16:819-825. See, also, Bolhassani, A. et al, Invitro and in vivo delivery of therapeutic proteins using cellpenetrating peptides. Peptides (November 2016), 87:50-63, whichdiscusses useful CPPs for the present compositions and methods,including without limitation, covalent bonded CPPs, such as Poly-Argpeptides, Tat and VP22, df Tat, Cyclic CPPs, IMT-P8 (particularly usefulfor transdermal delivery), seven arginine (R7) and Streptolysin O(SLO)-mediated systems and elastin like polypeptide, CPP-adaptor system,1, 2-Benzisothiazolin-3-one (BIT) and Tat, activatable cell-penetratingpeptides, LDP12, M918, BR2, peptide for ocular delivery (POD), nativeprotein independent of R11-CPP, Poly-arginine/Tat and Tat-PTD amongothers. Also identified are non-covalent bonded CPPs such as Pep-1,CADY-2, R8 and azo-R8, Penetratin, HR9 and IR9 peptides and pVEC. All ofthese documents are incorporated by reference herein for detaileddescriptions of known CPPs and PTDs. It is also anticipated that novelPTD/CPPs will prove useful with the compositions described herein.

Exemplary protein transduction domains include a YopM proteintransduction domain, an SspH1 protein transduction domain, or an IpaHprotein transduction domain. A useful YopM protein transduction domaincan have an amino acid sequence as set forth in SEQ ID NO 5.

In some embodiments, a fusion protein comprising a set of pairedpeptides linked to a protein transduction domain can have an amino acidsequence is at least 85% identical to the sequence set forth in SEQ IDNO. 10, 13, 16, 19, 22, or 24. In some embodiments, paired peptidefusion protein can have an amino acid sequence is at least 90% identicalto the sequence set forth in SEQ ID NO. 10, 13, 16, 19, 22, or 24. Insome embodiments, paired peptide fusion protein can have an amino acidsequence is at least 95% identical to the sequence set forth in SEQ IDNO. 10, 13, 16, 19, 22, or 24. In some embodiments, paired peptidefusion protein can have an amino acid sequence is at least 99% identicalto the sequence set forth in SEQ ID NO. 10, 13, 16, 19, 22, or 24. Insome embodiments, the paired peptide fusion protein can have an aminoacid sequence as set forth in SEQ ID NOs. 10, 13, 16, 19, 22, or 24.

Polypeptides. We tend to use the term “protein” to refer to longer orlarger amino acid polymers, and we tend to use the term “polypeptide” torefer to shorter sequences or to a chain of amino acid residues within alarger molecule (e.g., within a fusion protein) or complex. Both terms,however, are meant to describe an entity of two or more subunit aminoacids, amino acid analogs, or other peptidomimetics, regardless ofpost-translational modification (e.g., amidation, phosphorylation orglycosylation). The subunits can be linked by peptide bonds or otherbonds such as, for example, dicysteine, ester or ether bonds. The terms“amino acid” and “amino acid residue” refer to natural and/or unnaturalor synthetic amino acids, which may be D- or L-form optical isomers.Full-length proteins, analogs, mutants, and fragments thereof areencompassed by this definition.

The amino acid sequence of the bacterial effector polypeptides disclosedherein can be identical to the wild-type sequences of appropriatecomponents. Alternatively, any of the components can contain mutationssuch as deletions, additions, or substitutions. All that is required isthat the variant bacterial effector polypeptide have at least 5% (e.g.,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or evenmore) of the ability of the bacterial effector polypeptide containingonly wild-type sequences to specifically bind the target. Substitutionswill preferably be conservative substitutions. Conservativesubstitutions typically include substitutions within the followinggroups: glycine and alanine; valine, isoleucine, and leucine; asparticacid and glutamic acid; asparagine, glutamine, serine and threonine;lysine, histidine and arginine; and phenylalanine and tyrosine.

Variant bacterial effector polypeptides, e.g., those having one or moreamino acid substitutions relative to a native bacterial effectorpolypeptide amino acid sequence, can be prepared and modified asdescribed herein. Amino acid substitutions can be made, in some cases,by selecting substitutions that do not differ significantly in theireffect on maintaining (a) the structure of the peptide backbone in thearea of the substitution, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Forexample, naturally occurring residues can be divided into groups basedon side-chain properties: (1) hydrophobic amino acids (norleucine,methionine, alanine, valine, leucine, and isoleucine); (2) neutralhydrophilic amino acids (cysteine, serine, and threonine); (3) acidicamino acids (aspartic acid and glutamic acid); (4) basic amino acids(asparagine, glutamine, histidine, lysine, and arginine); (5) aminoacids that influence chain orientation (glycine and proline); and (6)aromatic amino acids (tryptophan, tyrosine, and phenylalanine)Substitutions made within these groups can be considered conservativesubstitutions. Non-limiting examples of useful substitutions include,without limitation, substitution of valine for alanine, lysine forarginine, glutamine for asparagine, glutamic acid for aspartic acid,serine for cysteine, asparagine for glutamine, aspartic acid forglutamic acid, proline for glycine, arginine for histidine, leucine forisoleucine, isoleucine for leucine, arginine for lysine, leucine formethionine, leucine for phenyalanine, glycine for proline, threonine forserine, serine for threonine, tyrosine for tryptophan, phenylalanine fortyrosine, and/or leucine for valine. Variant bacterial effectorpolypeptides having conservative and/or non-conservative substitutions(e.g., with respect to any of SEQ ID NOS: 3, 89, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 79, 10, 13, 16, 19,22, or 24), as well as fragments of any of SEQ ID NOS: 3, 89, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 79,10, 13, 16, 19, 22, or 24, 80, 81, 82, 83, 84, 85, 86, 87, or 88,fragments of variants of any of SEQ ID NOS: 3, 89, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 79. 10, 13,16, 19, 22, or 24, 80, 81, 82, 83, 84, 85, 86, 87, or 88 andpolypeptides comprising any of SEQ ID NOS: 3, 89, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 79, 10, 13,16, 19, 22, or 24, 80, 81, 82, 83, 84, 85, 86, 87, or 88, variants orfragments of any of SEQ ID NOS: 3, 89, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 79, 10, 13, 16, 19, 22,or 24, 80, 81, 82, 83, 84, 85, 86, 87, or 88, or fragments of variantsof any of SEQ ID NOS: 3, 89, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, and 79, 10, 13, 16, 19, 22, or 24, 80,81, 82, 83, 84, 85, 86, 87, or 88, can be screened for biologicalactivity using suitable assays, including those described herein. Forexample, the activity of a bacterial effector polypeptide, for example,NLeE or a mutant or fragment thereof, can be evaluated in vitro byassaying for methylase activity or in cell based systems to characterizeits effect on cytokine release.

In some embodiments, a bacterial effector polypeptide can comprise anamino acid sequence as set forth in SEQ ID NOS: 3, 89, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 79, 10,13, 16, 19, 22, or 24, 80, 81, 82, 83, 84, 85, 86, 87, or 88, but with aparticular number of amino acid substitutions. For example, a bacterialeffector polypeptides can have the amino acid sequence of any one of SEQID NOS: 3, 89, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 79, 10, 13, 16, 19, 22, or 24, 80, 81, 82, 83, 84,85, 86, 87, or 88, but with one, two, three, four, or five amino acidsubstitutions.

In some embodiments, a bacterial effector polypeptide as provided hereincan include an amino acid sequence with at least 85% (e.g., 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%,98.5%, 99.0%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequenceidentity to a region of a reference bacterial effector polypeptidesequence (e.g., SEQ ID NOS: 3, 89, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 79, 10, 13, 16, 19, 22, or 24,80, 81, 82, 83, 84, 85, 86, 87, or 88). Methods of determining percentsequence identity are discussed below.

In some embodiments, a polypeptide provided herein can be asubstantially pure polypeptide. As used herein, the term “substantiallypure” with reference to a polypeptide means that the polypeptide issubstantially free of other polypeptides, lipids, carbohydrates, andnucleic acid with which it is naturally associated. Thus, asubstantially pure polypeptide is any polypeptide that is removed fromits natural environment and is at least 60 percent pure or is anychemically synthesized polypeptide. A substantially pure polypeptide canbe at least about 60, 65, 70, 75, 80, 85, 90, 95, or 99 percent pure.Typically, a substantially pure polypeptide will yield a single majorband on a non-reducing polyacrylamide gel.

A variety of methods can be used to to make a polypeptide including, forexample, expression by prokaryotic systems, expression by eukaryoticsystems, and chemical synthesis techniques. Exemplary methods forpolypeptide purification purificinclude, without limitation,fractionation, centrifugation, and chromatography, e.g., gel filtration,ion exchange chromatography, reverse-phase HPLC and immunoaffinitypurification.

A polypeptide can be modified by linkage to a polymer such aspolyethylene glycol (PEG), or by fusion to another polypeptide such asalbumin, for example. For example, one or more PEG moieties can beconjugated to a bacterial effector polypeptide or fusion protein vialysine residues. Linkage to PEG or another suitable polymer, or fusionto albumin or another suitable polypeptide can result in a modifiedbacterial effector polypeptide or fusion protein having an increasedhalf life as compared to an unmodified bacterial effector polypeptide orfusion protein. Without being bound by a particular mechanism, anincreased serum half life can result from reduced proteolyticdegradation, immune recognition, or cell scavanging of the modifiedbacterial effector polypeptide or fusion protein. Methods for modifyinga polypeptide by linkage to PEG (also referred to as “PEGylation”) orother polymers include those set forth in U.S. Pat. No. 6,884,780;Cataliotti et al. ((2007) Trends Cardiovasc. Med. 17:10-14; Veronese andMero (2008) BioDrugs 22:315-329; Miller et al. (2006) Bioconjugate Chem.17:267-274; and Veronese and Pasut (2005) Drug Discov. Today10:1451-1458, all of which are incorporated herein by reference in theirentirety. Methods for modifying a polypeptide by fusion to albumininclude those set forth in U.S. Patent Publication No. 20040086976, andWang et al. (2004) Pharm. Res. 21:2105-2111, both of which areincorporated herein by reference in their entirety.

Nucleic acids. We may use the terms “nucleic acid” and “polynucleotide”interchangeably to refer to both RNA and DNA, including cDNA, genomicDNA, synthetic DNA, and DNA (or RNA) containing nucleic acid analogs,any of which may encode a polypeptide of the invention and all of whichare encompassed by the invention. Polynucleotides can have essentiallyany three-dimensional structure. A nucleic acid can be double-strandedor single-stranded (i.e., a sense strand or an antisense strand).Non-limiting examples of polynucleotides include genes, gene fragments,exons, introns, messenger RNA (mRNA) and portions thereof, transfer RNA,ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers, as well as nucleic acid analogs. In the context of thepresent invention, nucleic acids can encode a bacterial effectorpolypeptide, paired peptide fusion protein, or construct comprising apaired peptide fusion protein linked to a protein transduction domain.

An “isolated” nucleic acid can be, for example, a naturally-occurringDNA molecule or a fragment thereof, provided that at least one of thenucleic acid sequences normally found immediately flanking that DNAmolecule in a naturally-occurring genome is removed or absent. Thus, anisolated nucleic acid includes, without limitation, a DNA molecule thatexists as a separate molecule, independent of other sequences (e.g., achemically synthesized nucleic acid, or a cDNA or genomic DNA fragmentproduced by the polymerase chain reaction (PCR) or restrictionendonuclease treatment). An isolated nucleic acid also refers to a DNAmolecule that is incorporated into a vector, an autonomously replicatingplasmid, a virus, or into the genomic DNA of a prokaryote or eukaryote.In addition, an isolated nucleic acid can include an engineered nucleicacid such as a DNA molecule that is part of a hybrid or fusion nucleicacid. A nucleic acid existing among many (e.g., dozens, or hundreds tomillions) of other nucleic acids within, for example, cDNA libraries orgenomic libraries, or gel slices containing a genomic DNA restrictiondigest, is not an isolated nucleic acid.

Isolated nucleic acid molecules can be produced by in several ways. Forexample, polymerase chain reaction (PCR) techniques can be used toobtain an isolated nucleic acid containing a nucleotide sequencedescribed herein, including nucleotide sequences encoding a polypeptidedescribed herein. PCR can be used to amplify specific sequences from DNAas well as RNA, including sequences from total genomic DNA or totalcellular RNA. Generally, sequence information from the ends of theregion of interest or beyond is employed to design oligonucleotideprimers that are identical or similar in sequence to opposite strands ofthe template to be amplified. Various PCR strategies also are availableby which site-specific nucleotide sequence modifications can beintroduced into a template nucleic acid.

Isolated nucleic acids also can be chemically synthesized, either as asingle nucleic acid molecule (e.g., using automated DNA synthesis in the3′ to 5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >50-100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector.

Two nucleic acids or the polypeptides they encode may be described ashaving a certain degree of identity to one another. For example, abacterial effector polypeptide and a biologically active variant thereofmay be described as exhibiting a certain degree of identity. Alignmentsmay be assembled by locating short sequences in the Protein InformationResearch (PIR) site (http://pir.georgetown.edu), followed by analysiswith the “short nearly identical sequences” Basic Local Alignment SearchTool (BLAST) algorithm on the NCBI website(http://www.ncbi.nlm.nih.gov/blast).

As used herein, the term “percent sequence identity” refers to thedegree of identity between any given query sequence and a subjectsequence. For example, a bacterial effector polypeptide disclosed hereincan be the query sequence and a fragment of a bacterial effectorpolypeptide can be the subject sequence. Similarly, a fragment ofbacterial effector polypeptide can be the query sequence and abiologically active variant thereof can be the subject sequence.

To determine sequence identity, a query nucleic acid or amino acidsequence can be aligned to one or more subject nucleic acid or aminoacid sequences, respectively, using the computer program ClustalW(version 1.83, default parameters), which allows alignments of nucleicacid or protein sequences to be carried out across their entire length(global alignment).

ClustalW calculates the best match between a query and one or moresubject sequences and aligns them so that identities, similarities anddifferences can be determined. Gaps of one or more residues can beinserted into a query sequence, a subject sequence, or both, to maximizesequence alignments. For fast pair wise alignment of nucleic acidsequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For multiple alignments of nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pair wisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The output is a sequencealignment that reflects the relationship between sequences. ClustalW canbe run, for example, at the Baylor College of Medicine Search Launchersite (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and atthe European Bioinformatics Institute site on the World Wide Web(ebi.ac.uk/clustalw).

To determine a percent identity between a query sequence and a subjectsequence, ClustalW divides the number of identities in the bestalignment by the number of residues compared (gap positions areexcluded), and multiplies the result by 100. The output is the percentidentity of the subject sequence with respect to the query sequence. Itis noted that the percent identity value can be rounded to the nearesttenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to78.2.

The nucleic acids and polypeptides described herein may be referred toas “exogenous”. The term “exogenous” indicates that the nucleic acid orpolypeptide is part of, or encoded by, a recombinant nucleic acidconstruct, or is not in its natural environment. For example, anexogenous nucleic acid can be a sequence from one species introducedinto another species, i.e., a heterologous nucleic acid. Typically, suchan exogenous nucleic acid is introduced into the other species via arecombinant nucleic acid construct. An exogenous nucleic acid can alsobe a sequence that is native to an organism and that has beenreintroduced into cells of that organism. An exogenous nucleic acid thatincludes a native sequence can often be distinguished from the naturallyoccurring sequence by the presence of non-natural sequences linked tothe exogenous nucleic acid, e.g., non-native regulatory sequencesflanking a native sequence in a recombinant nucleic acid construct. Inaddition, stably transformed exogenous nucleic acids typically areintegrated at positions other than the position where the nativesequence is found.

Nucleic acids of the invention, that is, nucleic acids having anucleotide sequence of any paired peptides fusion proteins andconstructs disclosed herein, can include nucleic acids sequences thatare at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 99%identical to the reference sequences disclosed herein.

A nucleic acid, i.e., an oligonucleotide (e.g., a probe or a primer)that is specific for a target nucleic acid will hybridize to the targetnucleic acid under suitable conditions. We may refer to hybridization orhybridizing as the process by which an oligonucleotide single strandanneals with a complementary strand through base pairing under definedhybridization conditions. It is a specific, i.e., non-random,interaction between two complementary polynucleotides. Hybridization andthe strength of hybridization (i.e., the strength of the associationbetween the nucleic acids) is influenced by such factors as the degreeof complementary between the nucleic acids, stringency of the conditionsinvolved, and the melting temperature (Tm) of the formed hybrid. Thehybridization products can be duplexes or triplexes formed with targetsin solution or on solid supports.

Vectors. Vectors containing nucleic acids such as those described hereinalso are provided. A “vector” is a replicon, such as a plasmid, phage,or cosmid, into which another DNA segment may be inserted so as to bringabout the replication of the inserted segment. Generally, a vector iscapable of replication when associated with the proper control elements.Suitable vector backbones include, for example, those routinely used inthe art such as plasmids, viruses, artificial chromosomes, BACs, YACs,or PACs. The term “vector” includes cloning and expression vectors, aswell as viral vectors and integrating vectors. An “expression vector” isa vector that includes a regulatory region. A wide variety ofhost/expression vector combinations may be used to express the nucleicacid sequences described herein. Suitable expression vectors include,without limitation, plasmids and viral vectors derived from, forexample, bacteriophage, baculoviruses, and retroviruses. Numerousvectors and expression systems are commercially available from suchcorporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.),Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies(Carlsbad, Calif.).

The vectors provided herein also can include, for example, origins ofreplication, scaffold attachment regions (SARs), and/or markers. Amarker gene can confer a selectable phenotype on a host cell. Forexample, a marker can confer biocide resistance, such as resistance toan antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin). Asnoted above, an expression vector can include a tag sequence designed tofacilitate manipulation or detection (e.g., purification orlocalization) of the expressed polypeptide. Tag sequences, such as greenfluorescent protein (GFP), glutathione S-transferase (GST),polyhistidine, c-myc, hemagglutinin, or Flag™ tag sequences typicallyare expressed as a fusion with the encoded polypeptide. Such tags can beinserted anywhere within the polypeptide, including at either thecarboxyl or amino terminus.

Additional expression vectors also can include, for example, segments ofchromosomal, non-chromosomal and synthetic DNA sequences. Suitablevectors include derivatives of SV40 and known bacterial plasmids, e.g.,E. coli plasmids col E1, pCR1, pBR322, pMal-C2, pET, pGEX, pMB9 andtheir derivatives, plasmids such as RP4; phage DNAs, e.g., the numerousderivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 andfilamentous single stranded phage DNA; yeast plasmids such as the 2pplasmid or derivatives thereof, vectors useful in eukaryotic cells, suchas vectors useful in insect or mammalian cells; vectors derived fromcombinations of plasmids and phage DNAs, such as plasmids that have beenmodified to employ phage DNA or other expression control sequences.

Yeast expression systems can also be used. For example, the non-fusionpYES2 vector (XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI,Kpn1, and HindIII cloning sites; Invitrogen) or the fusion pYESHisA, B,C (XbaI, SphI, ShoI, NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIIIcloning sites.

The vector can also include a regulatory region. The term “regulatoryregion” refers to nucleotide sequences that influence transcription ortranslation initiation and rate, and stability and/or mobility of atranscription or translation product. Regulatory regions include,without limitation, promoter sequences, enhancer sequences, responseelements, protein recognition sites, inducible elements, protein bindingsequences, 5′ and 3′ untranslated regions (UTRs), transcriptional startsites, termination sequences, polyadenylation sequences, nuclearlocalization signals, and introns.

As used herein, the term “operably linked” refers to positioning of aregulatory region and a sequence to be transcribed in a nucleic acid soas to influence transcription or translation of such a sequence. Forexample, to bring a coding sequence under the control of a promoter, thetranslation initiation site of the translational reading frame of thepolypeptide is typically positioned between one and about fiftynucleotides downstream of the promoter. A promoter can, however, bepositioned as much as about 5,000 nucleotides upstream of thetranslation initiation site or about 2,000 nucleotides upstream of thetranscription start site. A promoter typically comprises at least a core(basal) promoter. A promoter also may include at least one controlelement, such as an enhancer sequence, an upstream element or anupstream activation region (UAR). The choice of promoters to be includeddepends upon several factors, including, but not limited to, efficiency,selectability, inducibility, desired expression level, and cell- ortissue-preferential expression.

Vectors include, for example, viral vectors (such as adenoviruses(“Ad”), adeno-associated viruses (AAV), and vesicular stomatitis virus(VSV) and retroviruses), liposomes and other lipid-containing complexes,and other macromolecular complexes capable of mediating delivery of apolynucleotide to a host cell. Vectors can also comprise othercomponents or functionalities that further modulate gene delivery and/orgene expression, or that otherwise provide beneficial properties to thetargeted cells.

A “recombinant viral vector” refers to a viral vector comprising one ormore heterologous gene products or sequences. Since many viral vectorsexhibit size-constraints associated with packaging, the heterologousgene products or sequences are typically introduced by replacing one ormore portions of the viral genome. Such viruses may becomereplication-defective, requiring the deleted function(s) to be providedin trans during viral replication and encapsidation (by using, e.g., ahelper virus or a packaging cell line carrying gene products necessaryfor replication and/or encapsidation).

Suitable nucleic acid delivery systems include recombinant viral vector,typically sequence from at least one of an adenovirus,adenovirus-associated virus (AAV), helper-dependent adenovirus,retrovirus, or hemagglutinating virus of Japan-liposome (HVJ) complex.In such cases, the viral vector comprises a strong eukaryotic promoteroperably linked to the polynucleotide e.g., a cytomegalovirus (CMV)promoter. The recombinant viral vector can include one or more of thepolynucleotides therein, preferably about one polynucleotide. In someembodiments, the viral vector used in the invention methods has a pfu(plague forming units) of from about 10⁸ to about 5×10¹⁰ pfu. Inembodiments in which the polynucleotide is to be administered with anon-viral vector, use of between from about 0.1 nanograms to about 4000micrograms will often be useful e.g., about 1 nanogram to about 100micrograms.

Additional vectors include retroviral vectors such as Moloney murineleukemia viruses and HIV-based viruses. One HIV-based viral vectorcomprises at least two vectors wherein the gag and pol genes are from anHIV genome and the env gene is from another virus. DNA viral vectorsinclude pox vectors such as orthopox or avipox vectors, herpesvirusvectors such as a herpes simplex I virus (HSV) vector.

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors may be an indication for some inventionembodiments. The adenovirus vector results in a shorter term expression(e.g., less than about a month) than adeno-associated virus, in someembodiments, may exhibit much longer expression. The particular vectorchosen will depend upon the target cell and the condition being treated.The selection of appropriate promoters can readily be accomplished. Anexample of a suitable promoter is the 763-base-pair cytomegalovirus(CMV) promoter. Other suitable promoters which may be used for geneexpression include, but are not limited to, the Rous sarcoma virus(RSV), the SV40 early promoter region, the herpes thymidine kinasepromoter, the regulatory sequences of the metallothionein (MMT) gene,prokaryotic expression vectors such as the β-lactamase promoter, the tacpromoter, promoter elements from yeast or other fungi such as the Gal 4promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerolkinase) promoter, alkaline phosphatase promoter; and the animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells, insulin gene control regionwhich is active in pancreatic beta cells, immunoglobulin gene controlregion which is active in lymphoid cells, mouse mammary tumor viruscontrol region which is active in testicular, breast, lymphoid and mastcells, albumin gene control region which is active in liver,alpha-fetoprotein gene control region which is active in liver, alpha1-antitrypsin gene control region which is active in the liver,beta-globin gene control region which is active in myeloid cells, myelinbasic protein gene control region which is active in oligodendrocytecells in the brain, myosin light chain-2 gene control region which isactive in skeletal muscle, and gonadotropic releasing hormone genecontrol region which is active in the hypothalamus. Certain proteins canexpressed using their native promoter. Other elements that can enhanceexpression can also be included such as an enhancer or a system thatresults in high levels of expression such as a tat gene and tar element.This cassette can then be inserted into a vector, e.g., a plasmid vectorsuch as, pUC19, pUC118, pBR322, or other known plasmid vectors, thatincludes, for example, an E. coli origin of replication. The plasmidvector may also include a selectable marker such as the β-lactamase genefor ampicillin resistance, provided that the marker polypeptide does notadversely affect the metabolism of the organism being treated.

Pharmaceutical carriers. The compositions also include apharmaceutically acceptable carrier. We use the terms “pharmaceuticallyacceptable” (or “pharmacologically acceptable”) to refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal or a human, asappropriate. The term “pharmaceutically acceptable carrier,” as usedherein, includes any and all solvents, dispersion media, coatings,antibacterial, isotonic and absorption delaying agents, buffers,excipients, binders, lubricants, gels, surfactants and the like, thatmay be used as media for a pharmaceutically acceptable substance.

Thus, the invention also includes pharmaceutical compositions whichcontain, as the active ingredient, a fusion protein comprising a set ofpaired peptides or a fusion protein comprising a set of paired peptideslinked a protein transduction domain, in combination with one or morepharmaceutically acceptable carriers. An active ingredient can be acomposition comprising a set of paired peptides linked to a proteintransduction domain and wherein prepared peptides comprise a firstbacterial effector polypeptide linked to a second bacterial effectorpolypeptide. In some embodiments, the polypeptide compositions can besterilized using conventional sterilization techniques before or afterit is combined with the pharmaceutically acceptable carrier. In makingthe compositions of the invention, the polypeptide compositions aretypically mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier in the form of, for example, a capsule, tablet,sachet, paper, or other container. When the excipient serves as adiluent, it can be a solid, semisolid, or liquid material (e.g., normalsaline), which acts as a vehicle, carrier or medium for the activeingredient. Thus, the compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium),ointments, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders. As is known in theart, the type of diluent can vary depending upon the intended route ofadministration. The resulting compositions can include additionalagents, such as preservatives. The excipient or carrier is selected onthe basis of the mode and route of administration. Suitablepharmaceutical carriers, as well as pharmaceutical necessities for usein pharmaceutical formulations, are described in Remington'sPharmaceutical Sciences (E. W. Martin), a well-known reference text inthis field, and in the USP/NF (United States Pharmacopeia and theNational Formulary). Some examples of suitable excipients includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrup, and methyl cellulose. The formulations can additionally include:lubricating agents such as talc, magnesium stearate, and mineral oil;wetting agents; emulsifying and suspending agents; preserving agentssuch as methyl- and propylhydroxy-benzoates; sweetening agents; andflavoring agents. The pharmaceutical compositions can also be formulatedso as to provide quick, sustained or delayed release of the activeingredient after administration to the patient.

Pharmaceutically acceptable compositions for use in the present methods,including those in which the polypeptides are entrapped in a colloid fororal delivery, can be prepared according to standard techniques. Thepolypeptides can be dried and compacted by grinding or pulverizing andinserted into a capsule for oral administration. In some embodiments,the polypeptides can be combined one or more excipients, for example, adisintegrant, a filler, a glidant, or a preservative. Suitable capsulesinclude both hard shell capsules or soft-shelled capsules. Anylipid-based or polymer-based colloid may be used to form the capusule.Exemplary polymers useful for colloid preparations include gelatin,plant polysaccharides or their derivatives such as carrageenans andmodified forms of starch and cellulose, e.g., hypromellose. Optionally,other ingredients may be added to the gelling agent solution, forexample plasticizers such as glycerin and/or sorbitol to decrease thecapsule's hardness, coloring agents, preservatives, disintegrants,lubricants and surface treatment. In some embodiments, the capsule doesnot include gelatin. In other embodiments, the capsule does not includeplant polysaccharides or their derivatives.

Regardless of their original source or the manner in which they areobtained, the polypeptides of the invention can be formulated inaccordance with their use. These compositions can be prepared in amanner well known in the pharmaceutical art, and can be administered bya variety of routes, depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may be oralor topical (including ophthalmic and to mucous membranes includingintranasal, vaginal and rectal delivery). In some embodiments,administration can be pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal) or ocular. Methods for ocular delivery caninclude topical administration (eye drops), subconjunctival, periocularor intravitreal injection or introduction by balloon catheter orophthalmic inserts surgically placed in the conjunctival sac. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular administration. Parenteraladministration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquids,powders, and the like. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable.

The compositions can be formulated in a unit dosage form, each dosagecontaining, for example, from about 0.005 mg to about 2000 mg ofpolypeptides per daily dose. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient. For preparingsolid compositions such as tablets, the principal active ingredient ismixed with a pharmaceutical excipient to form a solid preformulationcomposition containing a homogeneous mixture of a compound of thepresent invention. When referring to these preformulation compositionsas homogeneous, the active ingredient is typically dispersed evenlythroughout the composition so that the composition can be readilysubdivided into equally effective unit dosage forms such as tablets,pills and capsules. This solid preformulation is then subdivided intounit dosage forms of the type described above containing from, forexample, 0.005 mg to about 1000 mg of the compositions of the presentinvention.

The compositions can be formulated in a unit dosage form, each dosagecontaining, for example, from about 0.1 mg to about 50 mg, from about0.1 mg to about 40 mg, from about 0.1 mg to about 20 mg, from about 0.1mg to about 10 mg, from about 0.2 mg to about 20 mg, from about 0.3 mgto about 15 mg, from about 0.4 mg to about 10 mg, from about 0.5 mg toabout 1 mg; from about 0.5 mg to about 100 mg, from about 0.5 mg toabout 50 mg, from about 0.5 mg to about 30 mg, from about 0.5 mg toabout 20 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg toabout 5 mg; from about 1 mg from to about 50 mg, from about 1 mg toabout 30 mg, from about 1 mg to about 20 mg, from about 1 mg to about 10mg, from about 1 mg to about 5 mg; from about 5 mg to about 50 mg, fromabout 5 mg to about 20 mg, from about 5 mg to about 10 mg; from about 10mg to about 100 mg, from about 20 mg to about 200 mg, from about 30 mgto about 150 mg, from about 40 mg to about 100 mg, from about 50 mg toabout 100 mg of the active ingredient.

In some embodiments, tablets or pills of the present invention can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer which serves to resist disintegration inthe stomach and permit the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the compositions of the present invention canbe incorporated for administration orally or by injection includeaqueous solutions, suitably flavored syrups, aqueous or oil suspensions,and flavored emulsions with edible oils such as cottonseed oil, sesameoil, coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

The proportion or concentration of the compositions of the invention ina pharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration.

Methods of Treatment

The compositions disclosed herein are generally and variously useful fortreatment of inflammatory disorders and conditions. Inflammatorydisorders and conditions encompass a wide range of disorders coveringmany different systems and organs, including the gastrointestinal tract,the skin, the lungs, and the musculoskeletal system. Exemplaryinflammatory disorders include inflammatory bowel disease, rheumatoidarthritis, allergy, asthma, autoimmune diseases, coeliac disease,glomerulonephritis, hepatitis, preperfusion injury and transplantrejection. An inflammatory disorder can be a gastrointestinal disorder,for example, inflammatory bowel disease, Crohn's disease, and theileocolitis, ileocecal, jeunoileitis, and gastroduodenal subtypesof-Crohn's disease, and ulcerative colitis and subtypes of ulcerativecolitis.

A subject is effectively treated whenever a clinically beneficial resultensues. This may mean, for example, a complete resolution of thesymptoms associated with an inflammatory disorder, a decrease in theseverity of the symptoms associated with an inflammatory disorder, or aslowing of the progression of symptoms associated with an inflammatorydisorder. These methods can further include the steps of a) identifyinga subject (e.g., a patient and, more specifically, a human patient) whohas an inflammatory disorder; and b) providing to the subject acomposition comprising a paired peptide composition disclosed herein ina physiologically acceptable carrier. An amount of such a compositionprovided to the subject that results in a complete resolution of thesymptoms associated with an inflammatory disorder, a decrease in theseverity of the symptoms associated with an inflammatory disorder, or aslowing of the progression of symptoms associated with an inflammatorydisorder considered a therapeutically effective amount. The presentmethods may also include a monitoring step to help optimize dosing andscheduling as well as predict outcome.

The methods disclosed herein can be applied to a wide range of species,e.g., humans, non-human primates (e.g., monkeys), horses, pigs, cows orother livestock, dogs, cats or other mammals kept as pets, rats, mice,or other laboratory animals. The compositions described herein areuseful in therapeutic compositions and regimens or for the manufactureof a medicament for use in treatment of conditions as described herein(e.g., inflammatory disorders and conditions.)

When formulated as pharmaceuticals, the compositions can be administeredto any part of the host's body for subsequent delivery to a target cell.A composition can be delivered to, without limitation, the brain, thecerebrospinal fluid, joints, nasal mucosa, blood, lungs, intestines,muscle tissues, skin, or the peritoneal cavity of a mammal. In terms ofroutes of delivery, a composition can be administered by intravenous,intracranial, intraperitoneal, intramuscular, subcutaneous,intramuscular, intrarectal, intravaginal, intrathecal, intratracheal,intradermal, or transdermal injection, by oral or nasal administration,or by gradual perfusion over time. In a further example, an aerosolpreparation of a composition can be given to a host by inhalation.

Regardless of how the compositions are formulated, the dosage requiredwill depend on the route of administration, the nature of theformulation, the nature of the subject's condition, e.g., agastrointestinal disorder or a skin disorder, the subject's size,weight, surface area, age, and sex, other drugs being administered, andthe judgment of the attending clinicians. Suitable dosages are in therange of 0.01-1,000 mg/kg. Some typical dose ranges are from about 1μg/kg to about 1 g/kg of body weight per day. In some embodiments, thedose range is from about 0.01 mg/kg to about 100 mg/kg of body weightper day. In some embodiments, the dose can be, for example, 1 mg/kg, 2mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 50 mg/kg or 100 mg/kg. The dosage islikely to depend on such variables as the type and extent of progressionof the disease or disorder, the overall health status of the particularpatient, the relative biological efficacy of the compound selected,formulation of the excipient, and its route of administration.

Effective doses can be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems. For example, in vitroanalysis of cytokine production by peripheral blood mononuclear cells(PBMCs) can be a useful for assaying pro- and anti-inflammatoryresponses, e.g., secretion of IL-1beta, IL-12, IL-4 or IL-10, IL-6,IL-23, and TNF-alpha. Compositions can also be analyzed for effects inanimal models, for example, IgA production, cytokine production byexplants of Peyer's patches, and dendritic cell and T-cell responses.

Wide variations in the needed dosage are to be expected in view of thevariety of cellular targets and the differing efficiencies of variousroutes of administration. Variations in these dosage levels can beadjusted using standard empirical routines for optimization, as is wellunderstood in the art. Administrations can be single or multiple (e.g.,2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold).Encapsulation of the compounds in a suitable delivery vehicle (e.g.,polymeric microparticles or implantable devices) may increase theefficiency of delivery.

The duration of treatment with any composition provided herein can beany length of time from as short as one day to as long as the life spanof the host (e.g., many years). For example, a composition can beadministered once a week (for, for example, 4 weeks to many months oryears); once a month (for example, three to twelve months or for manyyears); or once a year for a period of 5 years, ten years, or longer. Itis also noted that the frequency of treatment can be variable. Forexample, the present compositions can be administered once (or twice,three times, etc.) daily, weekly, monthly, or yearly.

Any method known to those in the art can be used to determine if aparticular response is induced. Clinical methods that can assess thedegree of a particular disease state can be used to determine if aresponse is induced. For example, a subject can be monitored forsymptomatic relief, e.g., relief from colic, diarrhea, constipation,nausea, vomiting, abdominal pain, cramping, heartburn, abdominaldistention, flatulence, or incontinence, dermatitis, redness, pain,swelling. Alternatively or in addition, serum markers, imagingtechniques, e.g., ultrasound, x-rays, and endoscopic methods can beused.

The compositions may also be administered in conjunction with othertherapeutic modalities. Other therapeutic modalities will vary accordingto the particular disorder, but can include, for example,anti-inflammatory agents, antibiotics and other dietary treatments,anti-diarrhea medications, anti-emetics, anti-cholinergic agents,Concurrent administration of two or more therapeutic agents does notrequire that the agents be administered at the same time or by the sameroute, as long as there is an overlap in the time period during whichthe agents are exerting their therapeutic effect. Simultaneous orsequential administration is contemplated, as is administration ondifferent days or weeks.

Articles of Manufacture

The invention also features kits for administering the compositions.Accordingly, packaged products (e.g., sterile containers containing oneor more of the compositions described herein and packaged for storage,shipment, or sale at concentrated or ready-to-use concentrations) andkits, are also within the scope of the invention. A product can includea container (e.g., a vial, jar, bottle, bag, microplate or beads)containing one or more compositions of the invention. In addition, anarticle of manufacture further may include, for example, packagingmaterials, instructions for use, syringes, delivery devices, buffers orother control reagents.

For example, the kit can include a measured amount of a compositioncomprising a set of paired peptides wherein the set of paired peptidescomprises a first bacterial effect or polypeptide went to a secondbacterial effector polypeptide and a protein transduction domain. Thecompounds, agents, and/or reagents can be packaged in a suitablecontainer. The kit can further comprise instructions for administeringthe compositions. For example, the kit can include: a fusion proteincomprising a protein transduction domain and a set of paired peptides.The kit can also include a buffering agent, a preservative, and/or aprotein stabilizing agent. Each component of the kit can be enclosedwithin an individual container and all of the various containers can bewithin a single package. The product may also include a legend (e.g., aprinted label or insert or other medium describing the product's use(e.g., an audio- or videotape or computer readable medium)). The legendcan be associated with the container (e.g., affixed to the container)and can describe the manner in which the reagents can be used. Thereagents can be ready for use (e.g., present in appropriate units), andmay include one or more additional adjuvants, carriers or otherdiluents.

Also included as effectors or effector proteins of the compositions andmethods described herein are functional equivalents of the proteinsdescribed above. By the term “functional equivalent” is meant any aminoacid sequence or modification thereof that has the same targeting andimmune suppressing function of the naturally occurring effector protein.In one embodiment, such functional equivalents can have modifications ofone or more amino acids from the known sequences. In one embodiment,such functional equivalents can be a smaller fragment of the knownsequences. In one embodiment, such functional equivalents can be aderivative of the naturally occurring sequences or be derived from otherthan human sources. In one embodiment, such functional equivalents canbe altered by chemical modification or be altered by recombinantproduction to be associated with sequences with which the effectorproteins are not associated in nature. Similarly, chemical or structuralchanges or fragments of the nucleic acid sequences that encode theeffector proteins are also considered functional equivalents herein.

As used herein, the term “construct” as described herein refers to achemically synthesized or genetically engineered assemblage thatcomprises one or more PTD/CPP associated with one or more effectorproteins and further optionally associated with one or more targetingmoieties. The construct can be in the form of a polypeptide or a nucleicacid molecule encoding the polypeptide.

As used herein the term(s) “cosmeceutically or pharmaceuticallyacceptable carrier, excipient or formulation” refer to the components ofa composition that provide a vehicle for delivery. For example, wherethe cosmeceutical or pharmaceutical product is a topical composition,the carrier or formulation can contain typical components such ascremes, saline, vitamins, oils that are normally found in cosmetic orpharmaceutical preparations for skin. See, for example, U.S. Pat. No.5,635,497 which discloses an oil-in-water, fatty cream composition fortopical administration comprising from 60 to 80 percent by weight offatty components, from 1.5 to 5 percent by weight of at least onenon-ionic, hydrophilic surfactant having an HLB of at least 14, about 6%of fatty alcohols and esters, a therapeutically effective amount of atleast one topically active therapeutic agent, and water, provided thetopically active therapeutic agent is not dithranol or its derivatives.See, also International Patent Publication No. WO2014/076642.Pharmaceutically or cosmetically acceptable excipients suitable for thecompositions described herein can be selected from plasticizers,disintegrants, glidants, coloring agents, lubricants, stabilizers,adsorbents, preservatives, delivery retarders and mixtures thereof. Sucha composition may contain a transepidermal or transdermal carrier agentconsisting of acidic electrolyzed water having a pH of 1.0 to 4.0 andcomprise clusters of water having 5 to 10 molecules of water percluster, and a polyacrylate. Other suitable formulations may includeoils, emollients, lotions for topical and transdermal applications alongwith buffered/aqueous and saline solutions. See, also, texts such asTopical Drug Delivery Formulation (eds. Osborne and Amann), 2000,publishers Taylor & Francis, Drugs and the Pharmaceutical SciencesSeries #42.

The term “CAGE” as used herein refers to deep eutectic syntheticsolvent, a choline-based oil that has antimicrobial activity, which hasbeen shown to penetrate deep into the dermis. CAGE is described byZakrewsky, M. et al, Adv. Healthcare Mater. (March 2016), 5, 1282-1289,incorporated by reference herein. CAGE has been shown to be useful intransdermal protein delivery, wherein the protein is carried 15-20 celllayers into model skin preparations. See, e.g., Banerjee, A. et al, Adv.Healthcare Mater. 2017, 1601411 DOI: 10.1002/adhm.201601411,incorporated by reference herein.

Still other pharmaceutical strategies to enhance dermal delivery ofpeptides or proteins including carrier peptides, signaling peptidesneurotransmitter-inhibiting peptides and enzyme-inhibiting peptides,include chemical and physical penetration enhancers, such as listed inTable 2 of Badenhorst, T. et al, Pharmaceutical Strategies for theTopical Dermal Delivery of Peptides/Proteins for Cosmetic andTherapeutic Applications. Austin Journal of Pharmacology andTherapeutics (2014), 2(6):10. Also as discussed in this document arecoupling with lipophilic moiety, such as lauric, palmitic and otheracids, using CPP conjugates, formulation with microemulsions,encapsulation in liposomal vesicles and use of lipid particles, as wellas combinations of these formulations.

The term “targeting moiety” refers to constructs useful in fusion withthe effector proteins and/or PTD/CPPs described herein to direct thefusion protein or a nucleic acid sequence encoding it to a specific cellor tissue type within the body. Alterations to the fusion constructsdescribed by the addition of amino acid segments to naturally occurringeffector or fusion effector sequence, which enables the protein to bindand to specifically target cells, tissues or other target or physiologiccompartment in the human body create “targeted-effectors” and “targetedeffector fusions”. Such targeting moieties include amino acid segmentsthat enhance the efficacy of the preparation by its ability to beactivated due to conditions in a specific compartment of the body couldbe cleaved, for instance by proteolytic cleavage, addition ofpost-translational modifications, or forming of PPIs with cell andtissue specific host proteins. Examples of tissue-specific targetingpeptides for this use include those described in Jung, E. et al.,Identification of tissue-specific targeting peptide. J Comput Aided MolDes (October 2012) 26:1267-1275, incorporated by reference herein.Targeting moieties can also be antibodies, antibody fragments, aptamers,amino acid sequences, nucleic acid sequences that are complementary toor capable of binding a complementary sequence on a cell or tissue orchemical moieties that have a three-dimensional structure that can fitinto a three-dimensional pocket on the targeted cell or tissue. Forexample, a targeting sequence can be a hormone, or fragment thereof,that targets or binds its naturally occurring cell surface receptor, ortissue specific markers, etc.

The term “polypeptide,” when used in singular or plural form, generallyrefers to a polymer of amino acids joined together by peptide bonds andmay include unmodified or naturally occurring amino acids or modified orunnatural amino acids. In certain embodiment, the term polypeptiderefers to a construct formed by multiple shorter peptides joineddirectly, or indirectly via linkers, to form a single peptide. In oneembodiment, as described herein, a polypeptide is formed by the fusionof a PTD or CPP and an effector protein. In another embodiment, asdescribed herein, a polypeptide is formed by the fusion of a PTDattached to an effector protein, wherein the effector protein is furtherattached to another effector protein (with or without its own PTD). Instill another embodiment, the polypeptide of this invention is formed bythe covalent association of a first PTD fused to a first effectorprotein, a linker followed by an additional (e.g., second, third,fourth, etc) effector protein. The term “first” is used only todistinguish among the effectors. In yet another embodiment, eacheffector protein of the polypeptide is associated with its own PTD orCPP. In yet another embodiment, only one PTD/CPP is present. In anotherembodiment, the single PTD/CPP is located at the N terminus of thepolypeptide. In yet another embodiment, multiple PTD/CPP are present inthe polypeptide, each located at the N terminus of its associatedeffector protein. In another embodiment, each PTD/CPP is separated fromits effector protein by a linker. In another embodiment, each effectorprotein is separated from each additional effector protein by a linker.The first and additional effector constructs in a single polypeptide mayoccur in any order.

In still another embodiment of a polypeptide as described herein, thePTD/CPP are fused directly to the effector protein and each effectorprotein is fused to each additional effector protein. In certainembodiments, the polypeptides contain two or more different effectorproteins, each having its own target. In certain embodiments, thepolypeptides contain two or more different effector proteins, eachtargeting related targets. In still further embodiments, eachpolypeptide is further associated with a targeting moiety to target thepolypeptide to a specific tissue or cell type, e.g., skin, epidermis,dermis.

By “homologous protein” is meant a protein having a percent sequencesimilarity or identity of greater than 80%, greater than 85%, greaterthan 90%, greater than 95%, greater than 97%, or greater than 99% andsharing the same function as the effector protein.

As used herein, the term “polynucleotide,” when used in singular orplural form, generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA that encode any of the polypeptide constructs as describedabove. Thus, for instance, polynucleotides as defined herein include,without limitation, single- and double-stranded DNA, DNA includingsingle- and double-stranded regions, single- and double-stranded RNA,and RNA including single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or include single- and double-stranded regions. Inaddition, the term “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Theterm “polynucleotide” specifically includes cDNAs. The term includesDNAs (including cDNAs) and RNAs that contain one or more modified bases.In general, the term “polynucleotide” embraces all chemically,enzymatically and/or metabolically modified forms of unmodifiedpolynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells.

By “nucleic acid molecule” as used herein is meant the nucleic acidsequence that encodes a construct or polypeptide as described above. Thenucleic acid molecule can include other operative components, such asregulatory sequences directing expression of the construct orpolypeptide in a cell in vivo or in vitro. The nucleic acid molecule canbe in a vector.

By “vector” is meant an entity that delivers the nucleic acid moleculeto cells, for therapeutic or cosmetic purposes. As used herein, a vectormay include any genetic element including, without limitation, nakedDNA, a phage, transposon, cosmid, episome, plasmid, or a virus. Vectorsare generated using the techniques and sequences provided herein, inconjunction with techniques known to those of skill in the art. Suchtechniques include conventional cloning techniques of cDNA such as thosedescribed in texts such as Sambrook et al, Molecular Cloning: ALaboratory Manual, 3rd edition, 2001 Cold Spring Harbor Press, ColdSpring Harbor, N.Y., and current editions thereof, use of overlappingoligonucleotide sequences of the adenovirus genomes, polymerase chainreaction, CRISPR, gene editing, and any suitable method which providesthe desired nucleotide sequence.

By the term “attachment” or “attach” as used herein to describe theinteraction between the components of the constructs is meant covalentattachments or a variety of non-covalent types of attachment. Stillanother useful attachment mechanism involves via “affinityinteractions”, i.e., one domain fused to an antibody fragment thatrecognizes an epitope on the second domain to be used instead of the twodomains fused together. Other attachment chemistries useful inassembling the constructs described herein include, but are not limitedto, thiol-maleimide, thiol-haloacetate, amine-NHS, amine-isothiocyanate, azide-alkyne (CuAAC), tetrazole-cyclooctene (iEDDA).

The “linker” refers to any moiety used to attach or associate differentelements of the polypeptide//polynucleotide sequence components of theconstructs (i.e., the effector, the PTD, the targeting moiety) to eachother. Thus in one embodiment, the linker is a covalent bond. In anotherembodiment, the linker is a non-covalent bond. In an embodiment of apolynucleotide described herein, the linker is composed of at least oneto about 20 nucleic acids. Thus, in various embodiments, the linker isformed of a sequence of at least 3, 6, 9, 12, 15, 18, 21, 24, 27, 30,33, 36, 39, 42, 45, 48, 51, 54, 57 up to about 60 nucleic acids. In yetanother embodiment of a polypeptide as described herein, the linkerrefers to at least one to about 20 amino acids. Thus, in variousembodiments, the linker is formed of a sequence of at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 aminoacids. In still other embodiments, the linker can be a larger compoundor two or more compounds that associate covalently or non-covalently. Instill other embodiment, the linker can be a combination of the linkersdefined herein. The linkers used in the constructs of the compositionsand methods are in one embodiment chemically or enzymatically cleavable,such as by redox, pH, and the like. The linkers used in the constructsof the compositions and methods are in one embodiment non-cleavable.

The term “nucleic acid molecule” refers to a recombinant assemblednucleic acid sequence encoding a construct or polypeptide describedherein. The nucleic acid molecule may be naked DNA or RNA. Alternativelythe nucleic acid molecule may be associated operatively with regulatorysequences permitting expression of the construct or polypeptide in vivoor in vitro. The nucleic acid molecule may be a vector, plasmid vector,or be presented in a viral vector for delivery to the subject.Generation of such nucleic acid molecules with resort to the teachingsof this specification can utilize known recombinant and geneticengineering techniques. See, e.g., “Inflammatory conditions” as usedherein refer, in one aspect to inflammatory skin diseases, which are themost common problem in dermatology and cause pain, redness, swelling andthe sensation of heat. Such inflammatory skin conditions include,without limitation, non-specific rashes accompanied by skin itching andredness, sunburn, dermatitis, eczema, rosacea, seborrheic dermatitis,psoriasis, infection, skin injury or wounds, autoimmunity or agingeffects. Other inflammatory conditions (non-skin) include, withoutlimitation, autoimmune conditions, asthma, chronic peptic ulcer,tuberculosis, rheumatoid arthritis, periodontitis, ulcerative colitisand Crohn's disease, sinusitis, active hepatitis, gut dysbiosissyndromes and any other disease caused by NFkB/JNK/p38 pathwayactivation.

“Patient” or “subject” as used herein means a mammalian animal,including a human, a veterinary or farm animal, a domestic animal orpet, and animals normally used for clinical research. In one embodiment,the subject of these methods and compositions is a human.

The terms “a” or “an” refers to one or more. For example, “an expressioncassette” is understood to represent one or more such cassettes. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” areused interchangeably herein.

As used herein, the term “about” means a variability of plus or minus10% from the reference given, unless otherwise specified.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively, i.e., to include otherunspecified components or process steps. The words “consist”,“consisting”, and its variants, are to be interpreted exclusively,rather than inclusively, i.e., to exclude components or steps notspecifically recited.

With resort to the definitions of the components above, in one aspect,the inventors provide a composition comprising a first constructcomprising a selected immunomodulatory effector protein or itsfunctional equivalent thereof that targets a first functional domainoptionally linked covalently or non-covalently to a selected proteintransduction domain (PTD) or penetrating peptide (CPP); an additionalconstruct comprising a different effector protein or a functionalequivalent thereof that targets an additional functional domain,optionally linked to the same PTD or CPP of (a) or to an additional PTDor CPP, or a combination of a first and one or more additionalconstructs.

In one embodiment, the first and additional constructs are furtherlinked with a targeting moiety to direct activity of the composition toa specific cell or tissue, e.g., skin. In another embodiment, theconstructs are admixed in a pharmaceutically or cosmeceuticallyacceptable carrier or excipient or formulation, such as a formulationsuitable for topical administration to the skin.

One embodiment of such a composition is a single polypeptide comprisinga fusion of two or more effectors. Another embodiment is a singlepolypeptide comprising a first effector-PTD fused construct linkedcovalently or non-covalently to one or more of additional constructs.The polypeptide in a further embodiment contains one or more optionallinker amino acid sequences interposed between each construct of thepolypeptide. In one embodiment, the polypeptide contains fused to thefirst construct and/or additional construct, a targeting moiety todirect the polypeptide to a specific cell or tissue. In a furtherembodiment, the single polypeptide is in a pharmaceutically orcosmeceutically acceptable carrier or excipient or formulation.

Whether the constructs are admixed in a composition or present in one ormore polypeptides, the effector can be one or more of the effectorsidentified above, particularly in Tables 1 or 2. In one embodiment ofthe admixture or polypeptide, the first effector protein is NleE or afunctional equivalent thereof. In yet other embodiment, the additionaleffector protein is one or more of NleC, NleD, NleB, NleH, YopM, YopE,YopH, YopJ, YopP, SspH1, OspG, OspF, IpaH9.8, IpaH1.4, IpaH2.5, IpaH4.5,IpaH7.8 and SIrP. In other embodiments, the admixture of constructs inthe composition or present in the single polypeptide can be selectedfrom the following embodiments. In one embodiment, the first effector isNleE and the additional effector is NleC. In another embodiment, thefirst effector is NleE and the additional effector is NleD. In anotherembodiment, the first effector is NleE and the additional effector isNleB. In another embodiment, the first effector is NleE and theadditional effector is NleH. In another embodiment, the first effectoris NleE and the additional effector is YopB. In another embodiment, thefirst effector is NleE and the additional effector is YopH. In anotherembodiment, the first effector is NleE and the additional effector isYopJ. In another embodiment, the first effector is NleE and theadditional effector is YopP. In still another embodiment, the firsteffector is NleE and the additional effector is SspH1. In a furtherembodiment, the first effector is NleE and the additional effector isOspG. In another embodiment, the first effector is NleE and theadditional effector is OspF. In another embodiment, the first effectoris NleE and the additional effector is IpaH9.8. In still a furtherembodiment, the first effector is NleE and the additional effector isIpaH1.4. In another embodiment, the first effector is NleE and theadditional effector is IpaH2.5. In another embodiment, the firsteffector is NleE and the additional effector is IpaH4.5. In anotherembodiment, the first effector is NleE and the additional effector isIpaH7.8. In yet a further embodiment, the first effector is NleE and theadditional effector is SIrP. In still other embodiments of theconstructs or polypeptides, three or more effectors can be delivered ina single mixture of constructs or single polypeptide. The first andadditional effectors or effector constructs may occur in any order inthe fusions or in the polypeptides described herein.

In the above-listed constructs and polypeptides, a suitable PTD or CPPlinked to the first construct and optionally to each additionalconstruct or polypeptide is the naturally occurring PTD of the selectedeffector protein, e.g., the YopM PTD (SEQ ID NO: 2; amino acids 1-50 ofYopM). See U.S. Pat. No. 8,840,901.

The PTD/CPP can also be a functional equivalent, e.g., a mutated ormodified version of a naturally occurring PTD, such as the naturallyoccurring PTD sequence of SspH1, or of Shigella IpaH protein or anyother of the effectors. The PTD useful in the constructs andpolypeptides of the compositions can be a completely novel sequencewhich is developed to transport the construct or polypeptide orcomposition across cell membranes.

In one specific embodiment, the first construct is the YopM PTD fused toNleE. In another specific embodiment, the first construct is the SspH1PTD fused to NleE. In still another specific embodiment, the firstconstruct is the IpaH PTD fused to NleE. In still other embodiments, theadditional construct comprises YopM, optionally associated with the YopMPTD. In still other embodiments of the single polypeptide, thepolypeptide further contains at least one linker sequence as definedabove interposed between the first and the one or more additionalconstructs. In still other embodiments of the constructs and singlepolypeptide, each construct or polypeptide can further contain a fusedtargeting moiety, such as a skin cell targeting peptide or other cell ortissue targeting peptide. Given the number of effectors, PTD/CPPs,linkers and targeting moieties identified herein and in the citedpublications, any number of constructs or polypeptides may be preparedaccording to the teachings contained herein.

In still other embodiments of the above-listed constructs andpolypeptides, the admixture of protein constructs or constructscontained on the single polypeptide is such that the targeted functionaldomain of each effector protein in the construct is expressed in thesame cell. In another embodiment, each construct in admixture or in thesingle polypeptide has a non-overlapping redundant role in inhibitingNFkB, JNK and p38 pathways when present in a mammalian cell.

As described above, any of the compositions described herein, whether amixture of constructs, a single polypeptide or nucleic acid moleculesencoding them, can be prepared in a formulation comprises ingredientssuitable for application to, and absorption through, the cells of theskin. One such formulation employs the above-described CAGE solvent.Other suitable formulations may include oils, emollients, lotions fortopical and transdermal applications along with buffered/aqueous andsaline solutions.

In yet another aspect, the compositions described herein include nucleicacid molecules comprising a nucleic acid sequence encoding a firstconstruct, an additional construct, or the single polypeptide of any oneof the above-described embodiments. Such nucleic acid molecules can alsocomprise operatively associated regulatory sequences, such as promoters,enhancer, etc (see, e.g., Sambrook et al) necessary to express theconstruct or polypeptide in a suitable cell. In certain embodiments, thenucleic acid molecule is naked DNA or RNA. In certain embodiments, themolecule is part of a plasmid or contained in a recombinant vector orvirus. Methods for generating such nucleic acid molecules are within theskill of the art given the teachings herein. The nucleic acid moleculesmay also be delivered in a cosmeceutically or pharmaceuticallyacceptable carrier or excipient or formulation. Such formulations aredescribed in detail in available texts as described above.

These constructs and/or polypeptides may be formulation into a widevariety of cosmeceutically or pharmaceutically acceptable carrier orexcipient or formulation for many different uses and routes ofadministration. While topical administration is preferred for uses onskin inflammation, it is contemplated that other conventional routes ofadministration will be used for treating other inflammatory conditions.In some embodiments, routes of administration include transdermal(including patch formulation), intra-dermal injection (includinginfusion and subcutaneous injection). Other pharmaceutically acceptableroutes of administration include, but are not limited to, systemicroutes, such as intraperitoneal, intravenous, intranasal, intramuscular,intratracheal, subcutaneous, epidural, and oral routes and otherparenteral routes of administration or intratumoral or intranodaladministration. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceutical compoundsor compositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.Routes of administration may be combined, if desired.

Other methods of delivery of the effectors include via an attenuatedbacterial strain which expresses a functional Type-Three SecretionSystem (TTSS)-expressing microorganism and is engineered to containnucleic acid sequences encoding at least one of the effectorimmuno-modulatory proteins or fusions or polypeptide, wherein amino acidsequences also code for functional TTSS secretion signal sequences.These include attenuated bacteria engineered or induced to shed outermembrane vesicles (OMV) or other type of exosome-like, bacterial orcell-derived vesicle containing the proteins described herein.

In still other embodiments, the compositions, constructs, fusions andpolypeptides and nucleic acid molecules described herein may be furthermanipulated by encapsulation in liposomes, micro particles,microcapsules, or in recombinant cells capable of expressing thecompound, receptor-mediated endocytosis construction of a nucleic acidas part of a retroviral or other vector, etc.

Still other embodiments of compositions are provided herein. In certainembodiments, a composition of single effectors, or fusions of multipleeffectors, or fusions of single effector-PTD, or fusions of multipleeffectors and one or more PTD, or single/multipleeffector(s)-PTD-targeting moiety fusions are prepared. The polypeptidelinker regions and other non-native peptide sequences are engineeredinto fusion-effectors and can contain active segments which can lead totheir enhanced ability to be purified in active form. In anotherembodiment, the effector fusions can be modified recombinantly orchemically, or by e.g., editing methodologies to provide alteredsubcellular localization, altered stability, or altered ability toparticipate in protein-protein interactions. In one embodiment, theeffector fusions are modified to include amino acid segments normallyused as tags for efficient purification of proteins from complexmixtures (e.g., 6HIS, GST, and Maltose BP).

In still a further specific embodiment, each individual effector proteinor multiple effector fusion has an amino acid sequence that isdeliberately altered by designed site directed mutagenesis. In oneembodiment, such mutagenesis alters the activity of the effector interms of its potency as an anti-inflammatory effector. In anotherembodiment, the altered effector amino acid sequence alters theeffector's specificity for a target, catalytic activity, antigenicity,stability, ability to bind substrate, and/or ability to work with othereffectors in a preparation. Such mutagenic techniques can alter anyother common property of an effector protein for the benefit of theefficacy of the resulting therapeutic preparation.

In another aspect, the compositions containing the effector fusions canlead to the inhibition of at least one or more of an NF-κB, JNK and p38mediated signal transduction pathway protein in selected cells andtissues. Such inhibition provides an anti-inflammatory response, ananti-apoptotic effect or a pro-apoptotic effect in a target cell.

Still other embodiments of compositions can be obtained by selection ofthe components as taught herein resulting in apharmaceutical/cosmeceutical preparation, containing active andtherapeutic levels of the effector fusions and constructs as describedherein for use in preventing, treating, or ameliorating animmune-related disorder whose pathology stems from aberrant activationof the NFkB, JNK or P38 pathways.

In still other aspects, methods for designing the various constructs andpolypeptides described herein can be made using techniques well-known inthe art of recombinant genetic engineering and manipulation of thenucleic acid techniques. Suitable techniques are known to those of skillin the art, and as provided by many of the publications incorporated byreference herein. Selection of the appropriate techniques to create thevarious embodiments will depend upon the effectors, PTDs, linkers,targeting moieties and other components as set out above. Molecularbiology strategies to alter the proteins to make them more stable,specific, catalytic and robust are known. Further such known techniquescan be used to optimize the design, construction and delivery ofeffector fusions based upon knowledge of the specific pathways andenzymes used by the cell in a selected inflammatory stimulus.

For example, in one embodiment the selected effectors are subject torandom, high throughput mutagenesis in the catalytic active sites or thesites of substrate binding. Selection of effectors of these mutagenizedpopulations for variants with novel substrate specificities, rates ofcatalysis and their combination with other known or mutagenizedeffectors in the fusions, constructs and polypeptides described hereincan result in compositions with novel signal transduction inhibitionfunctions.

Still other known and available methods can be employed for creatingrecombinant fusion effector genes/proteins and for their expression,purification, stabilization as single effectors or fusion effectors.Such methods include producing/expressing the effector protein oreffector fusion proteins from cloned genes in any suitable cell basedsystems, including, without limitation, bacteria, yeast, insect, ormammalian cells. Methods for purification of the resulting fusionconstructs resulting in highly active, stable proteins which retain theability to target their native substrate also employ known techniques.Additional techniques for producing compositions of this inventioninclude, without limitation, optimization of the effector preparation byincorporation into liposomes, PEGylation of the effector constructs orfusions or polypeptides. The effector constructs may be subjected tochemical derivatization to associate or conjugate the constructphysically or in admixture with additional entities. Such entitiesinclude without limitation, lipids, liposomes, other drugs, and cargomolecules including nucleic acids, polypeptides, organic molecules,small organic molecules, metals, nano-particles, viruses, modifiedviruses, viral vectors, antibodies and/or plasmids as targets forconjugation.

Yet further aspects provide a variety of methods of using thecompositions, fusions, constructs and polypeptides described above inmethods for treating cosmetic conditions, such as inflammation orirritation due to normal skin agent, to the treatment of otherinflammatory conditions, such as cancer, gut dysbiosis syndromes, andother conditions identified herein caused by NFkB/JNK/p38 pathwayactivation. For example one therapeutic use is to ameliorate theinflammation in the tumor microenvironment (TME) in any malignant ornon-malignant condition that involves activation of the NFkB/JNK/p38pathway.

The various compositions defined above may be employed therapeuticallyin the down regulation/normalization of the immune response elicited bythe ectopic or pathologic activation of NFkB, JNK and P38 pathways. Inone embodiment, such methods of treating or suppressing pathwayactivation can occur in subjects suffering from the consequences ofnormal, naturally occurring conditions such as aging associatedinflammation. In yet another method, the compositions are administeredfor treatment of numerous conditions wherein the three pathways areaberrantly activated resulting in an inflammatory response. Suchaberrant responses include without limitation, infection, wound healing,reactive dermatitis, auto-immune disease, and malignant or non-malignantproliferative disorder. These diseases, conditions and syndromes whereinthe three pathways are aberrantly activated also include, but are notlimited to diseases caused by autoimmunity of the patient, topicalinflammation, chronic inflammation, gastroenteritis, chronic gastritis,inflammatory bowel diseases (IBD), colitis ulcerosa, psoriasis, allergicreactions, Crohns disease, dysbiosis syndromes, cancer (includinggliobastoma) rheumatoid arthritis related bone diseases characterized bychanges in bone resorption, reduction and relief of the signs andsymptoms associated with treating inflammation, and/or suppression ofthe immune system.

These methods involve administering via a suitable route ofadministration (as described above) an amount of the composition (i.e.,the effector fusions, effector-PTD fusions, effector-PTD-Targetingfusions, and the other embodiments described above) in an amountsufficient to reduce activation of the desired pathway.

Desirably, the methods further involve, in one aspect, administering asuitable dose or doses of the fusion construct(s) in a therapeuticregimen to the subject in need thereof. In one embodiment, suchadministration can occur once or more prior to, simultaneously with, orafter any conventional additional treatment for the conditions. In oneembodiment, where the condition is an infection, the additionalcomponent is an antibiotic. In another embodiment, wherein the conditionis a cancer, the additional component is radiation or chemotherapy.Still other known “additional components” may be selected by one ofskill in the art or the attending physician depending on the conditionbeing treated and the physical status of the subject.

Methods for determining the timing of frequency of administration willinclude an assessment of subject's response to the first administrationof the composition. The dose is generally the lowest dose of thecomposition that is effective to suppress activation of the NFKB,JNK/p38 pathway. In still other embodiments, a suboptimal dose isdelivered in a continuous infusion or a slow release formulation. Thedosage required will depend primarily on factors such as the conditionbeing treated, the age, weight and health of the patient, and may thusvary among patients. In one embodiment, where the composition comprisesan admixture of peptide constructs or the single polypeptide, one suchdose is about 1 to 25 μM protein/polypeptide. In another embodiment, thedose is less than 10 μM protein/polypeptide. In still anotherembodiment, the dose is between 1 μM and 5 μM protein/polypeptide. Inanother embodiment, the suboptimal dose is less than 1 μMprotein/polypeptide.

When the composition is in the form of a nucleic acid or vector ornucleic acid molecule, it is administered in sufficient amounts totransduce the targeted cells and to provide sufficient levels of genetransfer and expression to reduce or inhibit activation of the NFKB,JNK/p38 pathway and provide a therapeutic benefit without undue adverseor with medically acceptable physiological effects, which can bedetermined by those skilled in the medical arts. Dosages of thesetherapeutic compositions will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectiveadult dosage of a viral vector or siRNA nanoparticle is generally in therange of from about 100 μL to about 100 mL of a carrier containingconcentrations of from about 1×10⁶ to about 1×10¹⁵ particles, about1×10¹¹ to 1×10¹³ particles, or about 1×10⁹ to 1×10¹² virus particles.

In still another aspect, methods for use of the compositions describedherein involve veterinary use for the treatment of inflammatoryconditions in animals, e.g., for auto-immune diseases, reactivedermatitis, pruritis, alopecia and any other skin ailment for which theunderlying pathogenesis involves alteration of NFKB, JNK/p38 pathway.Selection of administration routes, dosages and therapeutic regimens maybe selected by a veterinarian.

In one aspect, a composition in a pharmaceutically acceptable carrier orexcipient or formulation is provided. The composition includes: (a) afirst construct comprising a selected immunomodulatory effector proteinor functional equivalent thereof that targets a first functional domainoptionally linked covalently or non-covalently or by affinity to aselected protein transduction domain (PTD) or penetrating peptide (CPP);(b) an additional construct comprising a different effector protein or afunctional equivalent thereof that targets an additional functionaldomain, optionally linked to the same PTD or CPP of (a) or to anadditional PTD or CPP, or (c) a combination of constructs (a) and (b) inany order.

In one embodiment of the composition, the first construct is linkedcovalently or non-covalently to one or more of the additional constructsin a single polypeptide. Additionally, the composition further includesan optional linker amino acid sequence interposed between eachconstruct.

In one embodiment, the functional equivalent includes a chemically orrecombinantly modified amino acid sequence of the effector protein, or afragment of the naturally-occurring effector amino acid sequence, or ofthe derivative of said chemically or recombinantly modified amino acidsequence of the effector protein that shares the functional activity ofthe effector protein.

In one embodiment, the first effector protein is NleE or a functionalequivalent thereof.

In one embodiment, the additional effector protein is one or more ofNleC, NleD, NleB, NleH, YopM, YopE, YopH, YopJ, YopP, SspH1, OspG, OspF,IpaH9.8, IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8 and SIrP. In one embodiment,the PTD or CPP is the naturally occurring PTD of the selected effectorprotein. In one embodiment, the PTD is a sequence of YopM, of SspH1, orof Shigella IpaH protein. In one embodiment, the PTD is amino acids 1-50SEQ ID NO: 2.

In one embodiment, the PTD or CPP is one or more of Poly-Arg, Tat andVP22, df Tat, a cyclic CPPs, IMT-P8, seven arginine (R7) andStreptolysin 0 (SLO)-mediated systems, elastin like polypeptide,CPP-adaptor system, 1, 2-Benzisothiazolin-3-one (BIT) and Tat,activatable cell-penetrating peptides, LDP12, M918, BR2, POD, nativeprotein independent of R11-CPP, Poly-arginine/Tat and Tat-PTD, Pep-1,CADY-2, R8, azo-R8, Penetratin, HR9 and IR9 peptides, or pVEC.

In one embodiment, each targeted functional domain of each effectorprotein in the construct is expressed in the same cell. In oneembodiment, each construct has a non-overlapping redundant role ininhibiting NFkB, JNK and p38 pathways when present in a mammalian cell.In one embodiment, the formulation comprises ingredients suitable forapplication to, and absorption through, the cells of the skin. In oneembodiment, the formulation comprises CAGE solvent. In one embodiment,the first construct is the YopM PTD fused to NleE or the SspH1 PTD fusedto NleE or the IpaH PTD fused to NleE. In one embodiment, the additionalconstruct comprises YopM, optionally associated with the YopM PTD. Inone embodiment, the composition further includes at least one linkerbetween the first and additional constructs.

In one embodiment, the first effector is NleE and the additionaleffector is NleC. In another embodiment, the first effector is NleE andthe additional effector is NleD. In another embodiment, the firsteffector is NleE and the additional effector is NleB. In anotherembodiment, the first effector is NleE and the additional effector isNleH. In another embodiment, the first effector is NleE and theadditional effector is YopB. In another embodiment, the first effectoris NleE and the additional effector is YopH. In another embodiment, thefirst effector is NleE and the additional effector is YopJ. In anotherembodiment, the first effector is NleE and the additional effector isYopP. In another embodiment, the first effector is NleE and theadditional effector is SspH1. In another embodiment, the first effectoris NleE and the additional effector is OspG. In another embodiment, thefirst effector is NleE and the additional effector is OspF. In anotherembodiment, the first effector is NleE and the additional effector isIpaH9.8. In another embodiment, the first effector is NleE and theadditional effector is IpaH1.4. In another embodiment, the firsteffector is NleE and the additional effector is IpaH2.5. In anotherembodiment, the first effector is NleE and the additional effector isIpaH4.5. In another embodiment, the first effector is NleE and theadditional effector is IpaH7.8. In another embodiment, the firsteffector is NleE and the additional effector is SIrP. In any of theembodiments above, the constructs may be in any order.

In one embodiment, the composition further includes a skin celltargeting peptide.

In another aspect, a nucleic acid construct is provided. The nucleicacid sequence includes a nucleic acid sequence encoding the polypeptideof any of the compositions described above.

In one embodiment, the nucleic acid construct further includesregulatory sequences necessary to express the polypeptide in a suitablecell.

In one embodiment, the nucleic acid construct is a DNA, RNA, a plasmidor a recombinant vector or virus.

In one embodiment, the nucleic acid construct is in a pharmaceuticallyacceptable carrier or excipient or formulation.

In another aspect, a method is provided for treating, preventing orameliorating an NFkB, JNK or p38 mediated inflammatory disorder in asubject, or a subject at risk from developing such disorder. The methodincludes administering to the subject any of the compositions or nucleicacid constructs described above.

In one embodiment of the method, the composition is administered in anamount sufficient to down-regulate the innate inflammatory response in atargeted cell or tissue of the subject mediated by the pathologic ornon-pathologic activation of intracellular NFkB, JNK or P38 signaltransduction pathway.

In one embodiment, the method reduces symptoms, and inhibitsprogression, of damage to the subject's cells or tissue caused byinflammation.

In one embodiment of the method, a composition containing multipleconstructs targets multiple targeted functional domains that areexpressed in the same cell and each construct plays multiple,non-overlapping redundant roles in inhibiting NFkB, JNK and p38pathways.

In one embodiment of the method, the presence of multiple constructs inthe compositions produces a synergistic therapeutic effect.

EXAMPLES Example 1: Preparation of Recombinant Constructs

Sequences. The native YopM, NleE, OSPZ, and IpA7.8 nucleotide sequenceswere codon optimized for efficient bacterial expression of the encodedpolypeptides. The optimized nucleotide sequences were synthesized denovo as long single stranded nucleotide chains. The overlapping andcomplementary single strand DNA sequences were annealed and the mixturesubject to polymerize chain reaction (PCR) to generate full-lengthdouble-stranded (ds) DNA. The ds DNA was gel isolated and cloned intoplasmid pQE60. The sequence of the inserted DNA was confirmed bynucleotide sequence analysis.

TAT-NleE wild type and NleE Mutant R107A. Both Vector and Amplicon weredigested using BamH1 restriction enzyme overnight at 37° C. The Vectorwas then phosphatase treated for 1 hour then heat inactivated prior toligation. All material was run through agarose gels and gene cleanedusing a Qiagen gel extraction kit. Ligation reaction was performed at16° C. overnight, and half of the volume was used to transform the DNAinto XL-1 blue competent cells. Colonies from the subsequenttransformation were picked and mini-preps were performed using a QiagenMini-Prep Kit. Resulting DNA was digested using BamH1 for 1 hr at 37° C.to test for the presence of the insert. 675 bp Positive clones were thendigested using EcoR5 (internal to the insert) and external Hind3 sitefor 1 hr at 37° C. to test the orientation of the insert. Positiveorientation yielded a drop-out that was 175 bp in size. Positive cloneswere then transformed into S9-competent cells to test protein expressionrates by mini-induction. A clone was chosen from that group, and usedfor future maxi-induction purifications under native conditions. Aschematic of the construct (based upon the backbone plasmid pQE60available from Qiagen Inc, Catalogue number: #32903) is shown in FIG. 3.

TAT-Shigella OSPZ. Both Vector and Amplicon were digested using BamH1restriction enzyme overnight at 37*C. The Vector was then phosphatasetreated for 1 hr then the enzyme was heat inactivated prior to ligation.All material was electrophoresed through agarose gels and the DNAisolated from the gel using a Qiagen DNA gel extraction kit. Ligationreaction was performed at 16*C overnight, and half of the volume wasused to transform the DNA into XL-1 blue competent cells. Colonies fromthe subsequent transformation were picked and mini-preps were performedusing a Qiagen Mini-Prep Kit. Resulting DNA was digested using BamH137*C for 1 hr to test for the presence of the 900 bp insert. Positiveclones were then digested using EcoR5 (internal to the insert) and Hind3located after the HIS-tag 37*C 1 hr to test the orientation of theinsert. Positive orientation yielded a drop-out DNA fragment that was200 bp in size. Positive clones were then transformed into S9-competentcells to test protein expression rates by mini-induction. A clone waschosen from that group, and used for future maxi-induction of proteinsand purification of protein under native conditions. A schematic of theconstruct is shown in FIG. 4.

YopM PTD-NleE wild type and Mutant R107A. Both Vector and Amplicon weredigested using BamH1 restriction enzyme overnight at 37*C. The Vectorwas then phosphatase treated for 1 hr and heat inactivated prior toligation. All material was run through agarose gels and gene cleanedusing a Qiagen gel extraction kit. Ligation reaction was performed at16*C overnight, and half of the volume was used to transform the DNAinto XL-1 blue competent cells. Colonies from the subsequenttransformation were picked and mini-preps were performed using a QiagenMini-Prep Kit. Resulting DNA was digested using Barn H1 at 37*C for 1 hrto test for the presence of the insert. Positive clones were thendigested using Bgl2 (internal to the insert) and Bgl2 at 37*C for 1 hrto test the orientation of the insert. Positive orientation yielded adrop-out that was 275 bp in size. Positive clones were then transformedinto S9-competent cells to test protein expression rates bymini-induction. A clone was chosen from that group, and used for futuremaxi-induction purifications under native conditions. A schematic of theconstruct is shown in FIG. 5.

YopM PTD-NleE wild type and Mutant R107A, No Linker, PAPA Linker, andGSGS Linker. A QE60 vector containing a codon optimized YopM sequencewas purchased from Epoch Life Science Inc. The plasmid was digestedusing Cla1 (internal to YopM) and Bgl2 (located outside of the cloningsite). NleE was then digested using Cla1 and Bam H1. Note that afterligation, the BamH1/Bgl2 site on the vector was lost. Because the vectorwas opened at a site that was internal to YopM, the ligation of NleE(along with its mutant R107A counterpart) lead to the fusion of the twoinserts. This site is located downstream to the PTD, thus leaving itintact. Variations of this construct were made using either PAPA or GSGSlinkers. These were added to the beginning of NleE during PCR by havingthe bridges be part of the forward primer sequence. Screening of thesevarious constructs were done via PCR of mini-preps using NleE Cla1 FORand TNHis Barn REV primers designed for NleE. Positive clones were thensent for sequencing to verify the clones prior to transformation intoS9-cells for protein expression. A schematic of the construct is shownin FIG. 6.

Schematics of the constructs used are shown in FIG. 1 and FIG. 2.

Purification of recombinant polypeptides. The transformed SG13009[pREP4] were plated on an agar plate containing both ampicillin andkanamycin. A single colony was used to inoculate a 200 mL culture,shaking at 200 RPM at 37° C. overnight. A volume of the overnightculture was added to 1 L of 2YT medium containing ampicillin andkanamycin and grown as described above in an incubator shaker at 200 RPM37° C. to an OD 600 nm of 0.6-0.8. Then, 1 mL of 1M Isopropylβ-D-1-thiogalactopyranoside (IPTG) was added to the culture and growthwas continued overnight with shaking at 200 RPM at 20° C. The followingmorning, cells were collected by centrifugation at 6,000 RPM at 4° C.The pelleted cells were resuspended in 40 mL Sonication Buffer andsonicated on ice for two cycles of 4-5 minutes each. The sonificationbuffer had a pH of 7.5 and 1 L of sonification buffer included onebottle of PBS, 300 mM NaCl, 10% Glycerol, 5 mM Imidazole. The remainderof the volume was filled with ddH₂O. The buffer was then filtered and 1ml 0.1M PMSF and 1 ml (2 mg/ml) each of Aprotinin, Leupeptine, andPepstatin were added fresh. The sonicate was centrifuged 12,000 RPM at4° C. for 30 minutes to separate lysed cells from soluble protein.

The supernatant was collected and incubated with pre-equilibrated Ni-NTAbeads (available from BioRad) for 1 hour at 4° C. by rotation. Thebinding capacity of polypeptides for Ni-NTA beads was about 5-10 mg/ml.Following the binding step, bound materials were centrifuged 4,000 RPM4° C. for 10 minutes to pellet the Ni-NTA beads and separate fromunwanted materials or “flow-through”. Beads were then washed twice in 40mL wash buffer by rotation in 4° C. for 30 minutes per wash cycle. Thewas buffer had a pH of 7.5 and 1 L of wash buffer included one bottle ofPBS, 300 mM NaCl, 10% Glycerol, and 20 mM Imidazole. The remainder ofthe volume was filled with ddH₂O In addition, 1 ml 0.1 M PMSF was addedfresh. The washed Ni-Beads were loaded on to a disposable gravity dripcolumn and washed with residual Wash Buffer.

Bound proteins were eluted with elution buffer, using 2× elution bufferper 1 mL of packed volume of beads, i.e., 2 ml elution buffer for 1 mlof Ni-NTA beads. The elution buffer had a pH of 7.5 and 100 ml ofelution buffer contained one bottle of PBS, 300 mM NaCl, 10% Glycerol,and 500 mM Imidazole. The remainder of the volume was filled with ddH₂O.The elution buffer was filtered before use. Eluate was collected in 2 mLfractions. The protein content of the fractions was analyzed usingBradford reagent. Individual fractions were dialyzed overnight indialysis buffer at 4° C. The dialysis buffer had a pH of 7.5 and 4 L ofelution buffer included one bottle of PBS, 300 mM NaCl, and 10%Glycerol. The remainder of the volume was then filled with ddH₂O. Thefollowing were freshly added to the elution buffer: 1 ml 0.1 M PMSF and1 ml 1 M DTT. In the morning, the dialysis buffer was discarded andreplaced with fresh buffer and dialysis was continued for an additional3 hours. After dialysis, the individual fractions were pooled andcentrifuged at 12,000 RPM at 4° C. for 20 minutes to remove anyprecipitation. The soluble protein was concentrated in the finalconcentration was determined using Bradford reagent.

Purified polypeptides were analyzed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) analysis using known volumes of bovine serumalbumin (BSA) as standards. Concentrated samples were aliquoted andstored at −80° C. Proteins were used directly after thawing on ice. AnSDS-PAGE analysis of purified recombinant chimeric effector proteins isshown in FIG. 7. Each lane contained 2 μg of protein. Proteins werevisualized by Coomassie blue staining.

Example 3: Cytokine Release

The effect of the bacterial effector proteins on cytokine release wasanalyzed in a cell-based assay using the human monocyte cell line,THP-1. Cytokine release was measured in vitro using the LEGENDplexMulti-Analyte Flow Assay Kit (BioLegend, Cat no. 740118) according tothe supplier's directions. For culturing THP-1 cells, RPMI withglutamine and 20% FBS was used. These cells were incubated at 37° C. inhumidified air 5% CO2 atmosphere. Finally, cell medium was changed every2-3 days, and the cells were passaged at 95% confluence. To allow thecells to adapt to their environment, 3×10⁵ cells were added to a 24-wellplate the night before. The next day, protein was added to these cellsfor a period of 3 hours. The protein was then washed out with PBS. Aftercells were stimulated overnight with LPS stimulation, a concentration of1 ug/mL was used. The next day, cells were washed again and cytokinewere measured. All 13 inflammatory cytokines were measured using theLEGENDplex Human Inflammation Plane (13-plex) assay from Biolegend.

We analyzed the effect of effector protein constructs on the release ofthe pro-inflammatory cytokines IL-1beta, TNF-alpha, 11-6, MCP-1, IL 23and IL-8. The effector protein constructs are shown in the table below:

Protein SEQ Construct transduction ID name domain Effector 1 LinkerEffector 2 NO YopM Full- YopM YopM 3 length TAT-NIeE TAT EPEC NLeE — —28 TAT Shigella TAT Shigella 34 OSPZ OSPZ YopM PTD- YopM EPEC NLeE — —37 NIeE YopM PTD- YopM YopM — EPEC NLeE 10 NIeE (No (L-rich) linker)YopM PTD- YopM YopM GSGS EPEC NLeE 16 NIeE (GSGS (L-rich) linker) YopMPTD- YopM YopM PAPA EPEC NLeE 13 NIeE (PAPA (L-rich) linker) YopM PTD-YopM YopM — — 7 YopM (L-rich) (L-rich) IpaH 7.8 IpaH 7.8 IpaH 9.8 IpaH9.8

The results of these experiments are shown in FIGS. 8, 9, 10, 11 and 12.As shown in FIGS. 8, 9, 10, 11 and 12, the effector proteins reduced thelevels of cytokines released into the medium relative to control samplesthat did not receive effector proteins. This effect was dose-dependent,with higher doses of effector proteins resulting in a steeper reductionin levels of released cytokines. These dose-dependent effects are shownin FIG. 8 with respect to IL-6. Similar dose-dependent effects wereobserved for IL-1beta, TNF-alpha, 11-6, MCP-1, IL 23, and IL-8.

The effector fusion proteins that included more than one effectorprotein domain produced a steeper reduction in the level of all releasedcytokines than did native effector proteins. As shown in FIG. 9, thepaired effector fusion proteins YopM PTD-YopM (L-rich)-NLeE; YopMPTD-YopM (L-rich)-GSGS-NLeE; YopM PTD-YopM (L-rich)-PAPA NLeE; and YopMPTD-YopM (L-rich) produced a greater reduction in levels of TNF alphareleased into the medium shown than did the full length YopM (rYopM) orShigella OSPZ. This reduction was observed for effector fusion proteinsthat did not include a linker sequence and for effector proteins thatincluded either a GSGS or a PAPA linker sequence.

As shown in FIG. 10, the paired effector fusion proteins YopM PTD-YopM(L-rich)-NLeE; YopM PTD-YopM (L-rich)-GSGS-NLeE; YopM PTD-YopM(L-rich)-PAPA NLeE; and YopM PTD-YopM (L-rich) produced a greaterreduction in levels of IL-6 released into the medium shown than did thefull length YopM (rYopM) or Shigella OSPZ. This reduction was observedfor effector fusion proteins that did not include a linker sequence andfor effector proteins that included either a GSGS or a PAPA linkersequence.

As shown in FIG. 11, the paired effector fusion proteins YopM PTD-YopM(L-rich)-NLeE; YopM PTD-YopM (L-rich)-GSGS-NLeE; YopM PTD-YopM(L-rich)-PAPA NLeE; and YopM PTD-YopM (L-rich) produced a greaterreduction in levels of MCP-1 released into the medium shown than did thefull length YopM (rYopM) or Shigella OSPZ. This reduction was observedfor effector fusion proteins that did not include a linker sequence andfor effector proteins that included either a GSGS or a PAPA linkersequence.

As shown in FIG. 12, the paired effector fusion proteins YopM PTD-YopM(L-rich)-NLeE; YopM PTD-YopM (L-rich)-GSGS-NLeE; YopM PTD-YopM(L-rich)-PAPA NLeE; and YopM PTD-YopM (L-rich) produced a greaterreduction in levels of IL-23 released into the medium shown then did thefull length YopM (rYopM) or Shigella OSPZ. This reduction was observedfor effector fusion proteins that did not include a linker sequence andfor effector proteins that included either a GSGS or a PAPA linkersequence.

Similar results were observed for IL-8. Taken together, the resultsshown in FIGS. 8, 9, 10, 11, and 12 indicate that fusion proteins thattarget multiple inflammatory pathways are more effective at reducing aninflammatory response than are proteins that target a singleinflammatory pathway.

Example 4: Caspase Activity

The effect of YopM on caspase activity was measured using R&D System'sCaspase-1/ICE Colorimetric Assay Kit (K111-100) according to thesupplier's instructions. THP-1. cells were incubated with rYopM or atruncated YopM (YopMo) and then stimulated with LPS (lipopolysaccharide)and ATP to induce activation of caspase 1. More specifically, 2×10⁶THP-1 cells were seeded in 6-well cell culture plates in triplicate. Thecells were incubated for 2 hours with rYopM (25 μg/ml) or a truncatedYopM (YopMo) and then LPS was added (1 μg/mL) and the cells wereincubated for an additional for 4 hours. Finally, ATP (5 mM) was addedand the cells were incubated for 45 minutes. To pellet the cells,centrifugation was carried out at 250×g for 10 min. The cell pellet(2×10⁶ cells) was resuspended in 50 μl lysis buffer (Caspase 1/ICEColorimetric Assay Kit R & D Systems) and incubated on ice for 10 min.Cell debris were pelleted at 10,000×g for 1 min. The colorimetric assaywas carried out in 96-well plates. For this purpose, 50 ul celllysate/well were placed and mixed with 50 ul 2× reaction buffer (Caspase1/ICE Colorimetric Assay Kit R & D Systems). After addition of 5 μl ofthe substrate Ac-YVAD-pNA, incubation was carried out at 37° C. for 3 h.Subsequently, the absorbance at 405 nm was measured.

The results of this experiment are shown in FIG. 13. As shown in FIG.13, a dose-dependent reduction of caspase 1 activity was observed inTHP-1 cells that had been treated with rYopM. A dose-dependent reductionof caspase 1 activity was also observed in THP-1 cells that had beentreated with the truncated YopM (YopMo). These data showed that thetruncated YopM (YopMo) that contained only the L-rich region of the YopMretained the caspase I reducing function of the full length YopM.

Examples 5: Effector Polypeptide Uptake

Confocal microscopy. Cell uptake of effector polypeptides was analyzedusing confocal microscopy. We analyzed single cell uptake of thefollowing fusion protein constructs:

Protein SEQ Construct transduction ID name domain Effector 1 LinkerEffector 2 NO TAT-NIeE TAT EPEC NLeE — — 28 YopM PTD- YopM YopM — EPECNLeE 10 NIeE (No (L-rich) linker) YopM PTD- YopM YopM GSGS EPEC NLeE 16NIeE (GSGS (L-rich) linker) YopM PTD- YopM YopM PAPA EPEC NLeE 13 NIeE(PAPA (L-rich) linker)

Fusion proteins were labeled with FITC using the Sigma-AldrichFluoroTag™ FITC Conjugation Kit according to the supplier's instructionsas follows. Protein and FITC were dissolved in carbonate-bicarbonatebuffer. The FITC was slowly added to the protein with stirring, thencovered with foil and stirred for two hours at room temperature. Theconjugate was separated from free FITC on a G-25 column in the fractionswere collected. Fractions containing conjugate were pooled and the F/Pratio of conjugate was determined spectrophotometrically. The labeledprotein was stabilized with 1% bovine serum albumin and 0.1% sodiumazide and stored at 0-5° C.

HaCat cells were cultured using DMEM High glucose medium with 10% FBS.These cells were incubated at 37° C. in humidified air 5% CO2atmosphere. Finally, cell medium was changed every 2-3 days, and thecells were passaged at 95% confluence. Cells were grown to appropriateconfluency. Next, appropriate coated coverslips were added to wellplates and the cells were seeded in the well plates containing thecoverslips. For uptake analysis, polypeptides were added to the cells toa final concentration of 50 ug/mL and incubated for two hours. Theprotein with the cells were incubated and the protein was aspiratedafter the incubation period was complete. The cells were fixed with 4%formaldehyde and washed once again. The membrane was permeabilized with0.2% triton-x-100 and washed again. The cells were blocked for 30minutes and incubated in primary antibody (either Cy5 or Actin)overnight. The antibody was then aspirated, washed, and incubated inappropriate secondary antibody for 30 minutes. The cells were thenwashed and DAPI solution was applied for 5 minutes. The cells were thenwashed and mounted using Prolong Gold Anti-Fade Reagent. The mountedcells were allowed to cure overnight and then imaged. The DAPI wasimaged using a wavelength of 490 nm. The Cy5 and Actin were imaged using594 nm wavelength.

Detection of protein from fixed cells was performed using Nikon 80iupright fluorescent microscope. A filter cube specific for the relativegreen range (˜488 nm) was used to detect FITC labelled protein withincells.

The results of this experiment are shown in FIGS. 14, 15, 16, and 17. Asshown in FIG. 14, incubation of cells with QE12-TAT-NleE resulted innuclear fluorescence. As shown in FIGS. 15 and 16, both QE60-YopMPTD-NleE (GSGS linker) and QE60-YopM PTD-NleE (PAPA linker),respectively, produced punctate cytoplasmic fluorescence that was notobserved in untreated control cells (FIG. 17). Taken together, thesedata show that the fusion polypeptides were taken up by individualcells.

Inverted microscopy. Uptake of QE12-TAT-NleE was analyzed by invertedmicroscopy as follows. A segment of skin was isolated from a shavedsection of a mouse, and the tissue was preserved in a saline solution tomaintain the cell viability within the tissue. The tissue was stainedwith 50 ug of FITC labeled TAT-NleE that was applied to the top of thesample and allowed to diffuse over a 2 hour period. Afterwards, thetissue was fixed with paraformaldehyde and stained with DAPI. The skinsegment was then sliced in 10 uM increments, and the top slice wasobserved in an inverted microscope was used with varying objectives. Asshown in FIG. 18, the TAT-NleE fusion polypeptide entered intact mouseskin and penetrated through multiple cell layers.

Two-photon microscopy. Penetration of TAT-NleE-into mouse skin wasanalyzed by two-photon microscopy. A 1 cm×1 cm segment of mouse skin wasstained with 50 μg of FITC-labeled TAT-NleE for two hours and counterstained with DAPI. the sample was analyzed by two-photon microscopy.More specifically, mouse hair was removed post-euthanasia and a 1 cm×1cm segment of skin was removed and submerged in 1×PBS pH 7.4 to maintaintissue viability. Afterwards, the PBS was removed, and 50 ugFITC-TAT-NleE was applied to the top of the skin and allowed to incubateat room temperature for two hours. Afterwards, the tissue was washedthree times for five minutes each with 1×PBS pH 7.4 to remove all waste.From there the tissue was permeabilized with Triton-x and fixed with 4%PFA. DAPI was then added to solution of PBS at a concentration of 1:1000and incubated in the tissue for 3-5 minutes. Then the washing period wasrepeated (three times for five minutes each in 1 ml of PBS). The tissuewas then mounted on a dish submerged in PBS ready for 2-photon imaging.

A 3 dimensional image is shown in FIG. 19 and a 10 uM slice of thestained tissue is shown in FIG. 20. As shown in FIGS. 19 and 20, theTAT-NleE penetrated the skin into the epidermis, but not the dermis.

Example 6: Methylase Activity

Methylase activity of recombinant proteins that included NleE effectorswas analyzed using the MTase glo assay from Promega. We assayed themethylase activity of wild-type NleE, mutant NleE R107A and fusionproteins YopM PTD-NleE (GSGS linker), YopM PTD-NleE (PAPA linker). Wealso analyzed the methylase activity of NLeE from Shigella.

The in vitro methylase assay was developed using as a base, the MTaseglo assay from Promega Inc. The primary assay buffer for enzyme activitywas (25 mM Tris, pH 8, 50 mM NaCl, 1 mM EDTA, 3 mM MgCL2, 0.1% BSA,0.005% Tween20, 5 mM DTT). To determine the effect of various vehiclesand buffers on NLeE a 40 uL aliquot of vehicle was spiked with 1 uL of20 mg/mL NLeE (0.5 mg/mL final). After ˜30 mins, 5 uL of spiked vehiclewas diluted into 5000 uL of primary assay buffer—(termed ‘SPIKED’ ingraphs). To determine the effect of diluted vehicle on NLeE activity, 5uL of vehicle diluted to 5000 uL of assay buffer was used. 1000 uL wasthen removed and used for the no enzyme controls. 1 uL of 0.5 mg/mL NLEEwas then added to 1000 uL of diluted vehicle and the assay commenced.The methyltransferase assay itself was performed in 4×1:2 serialdilutions of each sample generated above in assay buffer and 2.5 uLadded to 384-well assay plate. Reactions were initiated at various timepoints by the addition of 2.5 uL of 2× MTaseGlo, 1 uM GST-TAB2 proteinsubstrate and 20 uM SAM in assay buffer. After the time course,reactions were terminated by the addition of 5 uL MethyltransferaseDetection Reagent and the multiwell plates read for quantitative O.D.using a standard plate reader.

The results of this analysis are shown in FIGS. 21, 22 and 23. As shownin FIG. 21, both fusion constructs retained the methylase functionalityof native NLeE. As shown in FIG. 22, the presence of an R107A mutationin NLeE abolished methylase activity. As shown in FIG. 23, the ShigellaNleE showed methylase activity at levels comparable to that seen for theEPEC NleE

Example 7: Effect of Formulations on Nlee Methylation Activity

We analyzed the effective three different commercialformulations/delivery agents on NleE methylation activity in vitro.These were designated: Formula A, Formula B, and Formula C

The experimental setup was exactly as described above except that thebuffer controls contained an equivalent amount of Formula A, Formula Bor Formula C as controls. The results of this experiment are summarizedin FIG. 24. As shown in FIG. 24a , NLeE methylase activity was retainedin the presence of all three formulations. As shown in FIG. 24b , NLeEwas stable in the presence of each of the formulations, although thedegree of stability varied.

Example 8: How Specific is NleE for Inhibiting NFKB?

We cloned expressed and tested 64 other human C4 ZF containing proteins,among them the sequences identified in the table below. But few of themwere modified by NleE. Thus, the specificity of NleE is extremely high.

CAN15_HUMAN/7-26 WsCvr..CtflNpagqrqCsiC CAN15_HUMAN/48-67WpCar..CtfrNflgkeaCevC CAN15_HUMAN/147-166 WaCpr..CtlhNtpvassCsvCCAN15_HUMAN/344-363 WsCak..CtlrNptvaprCsaC CAN15_HUMAN/416-435WaCpa..CtllNalrakhCaaC EWS_HUMAN/522-543 WqCpnpgCgnqNfawrteCnqCOncogene RNA Binder FUS_HUMAN/426-447 WkCpnptCenmNfswrneCnqCOncogene Annealing HOIL1_HUMAN/197-216 WqCpg..CtfiNkptrpgCemCBinds Ub Chains MDM2_HUMAN/303-322 WkCts..CnemNpplpshCnrC P53 regulationMDM4_HUMAN/304-323 WqCte..CkkfNspskryCfrC P53 regulationNEIL3_HUMAN/321-340 WtCvv..CtliNkpsskaCdaC Endonuclease8 RepairNPL4_HUMAN/584-603 WaCqh..CtfmNqpgtghCemC Ub Chaperone EradNRP1_YEAST/359-378 WnCps..CgfsNfqrrtaCfrC Rna Binder No FcnNRP1_YEAST/585-604 WkCst..CtyhNfaknvvClrC NU153_HUMAN/662-681WqCdt..CllqNkvtdnkCiaC NUP153 Nuc Pore NU153_HUMAN/726-745WdCdt..ClvqNkpeaikCvaC NU153_HUMAN/797-816 WeCsv..CcvsNnaednkCvsCNU153_HUMAN/855-874 WdCel..ClvqNkadstkClaC RBM10_HUMAN/217-236WlCnk..CgvqNfkrrekCfkC RNA Binding RBM5_HUMAN/185-204WlCnk..CclnNfrkrlkCfrC RNA Binding RBP2_HUMAN/1356-1375WhCns..CslkNastakkCvsC RANBP2 Huge Nuc Pore RBP2_HUMAN/1419-1438WdCsi..ClvrNeptvsrCiaC RBP2_HUMAN/1483-1502 WdCsa..ClvqNegsstkCaaCRBP2_HUMAN/1547-1566 WdCss..ClvrNeanatrCvaC RBP2_HUMAN/1610-1629WdCsv..ClvrNeasatkCiaC RBP2_HUMAN/1669-1688 WdCsv..ClvrNeasatkCiaCRBP2_HUMAN/1728-1747 WdCsv..ClvrNeasatkCiaC RBP2_HUMAN/1785-1804WdCsv..CcvqNessslkCvaC RBP56 HUMAN/358-379 WvCpnpsCgnmNfarrnsCnqCRNF31 HUMAN/304-323 WhCaa..CamlNepwavlCvaC RNF31 HUMAN/354-373WaCqs..CtfeNeaaavlCsiC RNF31 HUMAN/413-432 WyCih..CtfcNsspgwvCvmCRYBP_HUMAN/25-44 WdCsv..CtfrNsaeafkCsiC SHRPN_HUMAN/352-371WsCps..CtfiNapdrpgCemC TAB2_HUMAN/668-687 WnCta..CtflNhpalirCeqCNFKB pathway TAB3_HUMAN/687-706 WnCds..CtflNhpalnrCeqC NFKB pathwayTX13A_HUMAN/380-399 WdCpw..CnavNfsrrdtCfdC

A cysteine methylation assay was performed and shows that NleE does notutilize zinc fingers of known highly related zinc fingers. NleE targetsNFKB signaling at a key step in TAK1 kinase activation. The CYS-4 ZincFinger of TAB2 was required to bind free K63 Ub chains to activate TAK1kinase, which thereby triggers NFKB signaling. The TAB2/3 Cys 4Zinc-Finger binds K63 linked Ub chains: The metal chelated structure ofthe ZF required binding. NleE targets NFKB signaling by methylating asingle Cysteine in the C4 ZF TAB2 of TAB2 abolishing K63 Ub sensing. Cys673 in TAB2 is the target of NleE. This highly specific activity of NleEis repurposed in the compositions described herein to shut down the NFKBpathway and reduce inflammation.

Example 9: Penetration of Recombinant NleE into the Skin in an Ex VivoPig Skin Model

A key claim and potential stumbling block to use of NleE as a skinanti-inflammatory compound is to show that this globular folded proteinof MW 38 kDa is able to penetrate the dermis and epidermis in order toaccess cells in which the NFKB system is aberrantly activated. Arecombinant construct was prepared by fusing the amino acid sequence ofNleE to an N-terminal histidine, and covalently labelling that peptidewith FITC dye, resulting in the peptide His-NleE—FITC.

This construct was mixed with the eutectic, cholinate-based solventCAGE, as described above at a concentration of 1-10 mg/ml. The amount ofpeptide in CAGE or PBS was 1 mg/ml. The final CAGE concentration was 90%v/v. The mixture was applied to live pig skin being perfused in a Franzdiffusion chamber at 37 degrees C. for 24 or 48 hours. The Franzdiffusion chamber is described by Technical Brief 2009 vol. 10, ParticleSciences—Drug Development Services (Bethlehem, Pa.). After these timeperiods, skin was harvested, fixed and sectioned followed by microscopyto visualize the FITC dye. The results (FIG. 4) show thatCAGE-solubilized NleE FITC was able penetrate the stratum corneum,epidermis and dermis and that virtually every cellular compartment ofthe skin. Up to 20-30 cell layers were contacted.

In comparison to published reports by Banerjee et. al 2017, particularlyFIG. 1 using CAGE to transport the globular proteins ovalbumin andbovine serum albumin, the NleE penetrates as well if not better thanthese test proteins. NleE solubilized in PBS and applied to the pig skinshowed no transduction past the stratum corneum.

In summary, this pre-clinical data provides support that recombinantNleE protein is stable in the eutectic solvent CAGE and can be deliveredto multi layers of the skin locally in a topical manner. It alsoprovides support for using NleE protein and the combinations describedherein that contain an intrinsic PTD domain which renders the proteincapable of penetrating cells and tissues and turning off inflammatorycytokines. The ability of these combinations of proteins to penetrate,in an apparently active form and inhibit production of a multitude ofinflammatory cytokines, is reflected in its therapeutic effect onpsoriatic lesions in a mouse skin model. These compositions delivered ina cream topically, in therapeutic levels are not anticipated toappreciably enter the regional lymphatics.

These observations strongly support the use of recombinant bacterialeffector proteins containing PTDs (Table1) are useful as a platform forinhibiting various accessible inflammatory disease processes in thebody.

Thus, methods and compositions for treating inflammation related toactivation of the NFkB, JNK and p38 pathways, e.g., inflammatoryconditions of skin, include a composition comprising a first constructcomprising a selected immunomodulatory effector protein or functionalequivalent thereof that targets a first functional domain; and at leastone additional construct comprising a different effector protein or afunctional equivalent thereof that targets an additional functionaldomain. The first construct is optionally linked to a selected proteintransduction domain (PTD) or penetrating peptide (CPP) or to a targetingmoiety. The additional construct is optionally linked to the same PTD orCPP of the first construct or to an additional PTD or CPP. In oneembodiment, a combination of these constructs is provided. In anotherembodiment the first construct is fused to one or more additionalconstructs in a single polypeptide. The composition further comprise anoptional linker amino acid sequence interposed between each constructand an optional targeting moiety.

Each and every patent, patent application, and publication, includingwebsites cited throughout specification are incorporated herein byreference. Similarly, the SEQ ID NOs which are reference herein andwhich appear in the appended Sequence Listing are incorporated byreference. While the invention has been described with reference toparticular embodiments, it will be appreciated that modifications can bemade without departing from the spirit of the invention. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. A composition comprising a set of paired peptides, wherein the set ofpaired peptides is linked to a protein transduction domain, and whereinthe set of paired peptides comprises a first bacterial effectorpolypeptide or fragment thereof linked to a second bacterial effectorpolypeptide or fragment thereof.
 2. The composition of claim 1, whereinthe first bacterial effector polypeptide or fragment thereof and secondbacterial effector polypeptide or fragment thereof are different.
 3. Thecomposition of claim 1, wherein a first bacterial effector polypeptideor fragment thereof and the second bacterial effector polypeptide orfragment thereof are immunomodulatory.
 4. The composition of claim 1,wherein the first bacterial effector polypeptide or fragment thereof andthe second bacterial effector polypeptide or fragment thereof recognizea different molecular target or modulate a different inflammatorypathway.
 5. The composition of claim 4, wherein the inflammatory pathwayis the NFkB pathway, the JNK pathway, the p38 pathway or the STINGpathway.
 6. The composition of claim 1, wherein the protein transductiondomain and the set of paired peptides comprise a fusion protein.
 7. Thefusion protein of claim 6, further comprising one or more linkers. 8.The fusion protein of claim 7, wherein the linker is positioned betweenthe first bacterial effector polypeptide or fragment thereof and thesecond bacterial effector polypeptide or fragment thereof.
 9. Thecomposition of claim 1, wherein the protein transduction domain is aYopM protein transduction domain, an SspH1 protein transduction domain,or an lpaH protein transduction domain.
 10. The composition of claim 9,wherein the protein transduction domain is a YopM protein transductiondomain.
 11. The composition of claim 10, wherein the proteintransduction domain comprises SECS ID NO.
 5. 12. The composition ofclaim 1, wherein the protein transduction domain is selected from thegroup consisting of Poly-Arg, Tat and VP22, df Tat, a cyclic CPPs,IMT-P8, seven arginine (R7) and Streptolysin 0 (SLO)-mediated systems,elastin like polypeptide, CPP-adaptor system, 1,2-Benzisothiazolin-3-one (BIT) and Tat, activatable cell-penetratingpeptides, LDP12, BR2, POD, native protein independent of R11-CPP,Poly-arginine/Tat and Tat-PTO, Pep-1, CADY-2, R8, azo-R8, Penetratin,HR9 and IR9 peptides, or pVEC.
 13. The composition of claim 1, whereinthe first bacterial effector polypeptide or fragment thereof is apolypeptide selected from the group consisting of NleE, NleC, NleD,NleB, NleH, YopM, YopE, YopH, YopJ, YopP, SspH1, OspG, OspF, lpaH9.8,lpaH1.4, lpaH2.5, lpaH4.5, lpaH7.8 and SlrP, and the second bacterialeffector polypeptide or fragment thereof is a polypeptide selected fromthe group consisting of NleE, NleC, NleD, NleB, NleH, YopM, YopE, YopH,YopJ, YopP, SspH1, OspG, OspF, lpaH9.8, lpaH1.4, lpaH2.5, lpaH4.5,lpaH7.8 and SlrP.
 14. The composition of claim 13, wherein the firstbacterial effector polypeptide or fragment thereof is a polypeptidehaving 90% sequence identity to an amino acid sequence set forth in thegroup consisting of SEQ ID NOs 3, 89, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 79 and the secondbacterial effector polypeptide or fragment thereof is a polypeptidehaving 90% sequence identity to an amino acid sequence set forth in thegroup consisting of SEQ ID NOs.3, 89, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and
 79. 15. The compositionof claim 1, wherein (a) the first bacterial effector polypeptide orfragment thereof is a YopM polypeptide or a fragment thereof or an NLeEpolypeptide or a fragment thereof; or (b) the second bacterial effectorpolypeptide or fragment thereof is a YopM polypeptide or a fragmentthereof or an NLeE polypeptide or a fragment thereof.
 16. (canceled) 17.The composition of claim 1, wherein the first bacterial effectorpolypeptide or fragment thereof is a YopM polypeptide or a fragmentthereof and the second bacterial effector polypeptide or fragmentthereof is an NLeE polypeptide or a fragment thereof.
 18. The fusionprotein of claim 6, wherein the amino acid sequence is at least 85%identical to the sequence set forth in SEQ ID NOs. 10, 13, 16, 19, 22,or
 24. 19-35. (canceled)
 36. A fusion protein comprising a set of pairedpeptides, wherein the set of paired peptides comprises a first bacterialeffector polypeptide or fragment thereof linked to a second bacterialeffector polypeptide or fragment thereof. 37-56. (canceled)
 57. Acomposition comprising a protein transduction domain polypeptide linkedto two or more bacterial effector polypeptides or fragments thereof. 58.(canceled)